Emulsions or microemulsions for use in endoscopic mucosal resectioning and/or endoscopic submucosal dissection

ABSTRACT

The present invention relates to a pharmaceutical composition in form of emulsion or microemulsion and the use thereof as aid during endoscopic procedures in which it is injected in a target tissue in order to form a cushion. More in details, the invention relates to a method for performing an endoscopic procedure, which comprises injecting said pharmaceutical composition in form of emulsion or microemulsion in a target tissue of a patient, in order to form a cushion, which cushion is then optionally subjected to an endoscopic surgical procedure, such as a resection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/154,857 filed 13 May 2016, which in turn is a continuationof U.S. patent application Ser. No. 14/950,525 filed 24 Nov. 2015, nowU.S. Pat. No. 9,364,580, which in turn is a division of U.S. patentapplication Ser. No. 14/546,925 filed 18 Nov. 2014, now U.S. Pat. No.9,226,996, which claims priority to Italian application No.MI2013A001924 filed 20 Nov. 2013. Each application is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition in form ofemulsion or microemulsion and the use thereof as aid during endoscopicprocedures in which it is injected in a target tissue in order to form acushion. More in details, the invention relates to a method forperforming an endoscopic procedure, which comprises injecting saidpharmaceutical composition in form of emulsion or microemulsion in atarget tissue of a patient, in order to form a cushion, which cushion isthen optionally subjected to an endoscopic surgical procedure, such as aresection.

BACKGROUND OF THE INVENTION

Endoscopy is a diagnostic and medical procedure which allows to examinethe interior of a hollow organ or cavity of the body by means of aninstrument called endoscope, without employing invasive surgery.Endoscopy is commonly used for diagnostic purposes, even though minor,non-invasive surgical and non-surgical interventions can be performedduring an endoscopic procedure. Typically, said minor interventionscomprise cauterization of a bleeding vessel, widening a narrowesophagus, removing polyps, adenomas and small tumors, performingbiopsies or removing a foreign object. Endoscopy is used by specialiststo examine, for example, the gastrointestinal tract, the respiratorytract, the ear, the urinary tract, the female reproductive system and,through small incisions, normally closed body cavities such as theabdominal or pelvic cavity (laparoscopy), the interior of a joint(arthroscopy) and organs of the chest (thoracoscopy andmediastinoscopy). The endoscope is an illuminated usually optic fiberflexible or rigid tubular instrument for visualizing the interior of ahollow organ or part (as the bladder, esophagus, stomach or intestine)for diagnostic or therapeutic purposes, that typically has one or moreworking channels to enable passage of instruments (such as forceps,electrosurgical knife, endoscopic injection needles or scissors) or tofacilitate the removal of bioptic samples. It includes a suitable lampand imaging device at its distal portion, and it can be inserted throughnatural occurring openings of the body, such as the mouth, the anus, theear, the nose or through small surgical incisions. Given the widevariety of body organs or cavities which can be examined by means ofendoscopic procedures, several types of endoscopes exist, such as, forexample, laryngoscope, thoracoscope, angioscope, colonoscope,enteroscope, sigmoidoscope, rectoscope, proctoscope, anoscope,arthroscope, rhinoscope, laparoscope, hysteroscope, encephaloscope,nephroscope, esophagoscope, bronchoscope, gastroscope, amnioscope,cystoscope.

In particular, endoscopic procedures are widely applied in thegastrointestinal tract, both for diagnostic purposes and for smallinterventions. With the progress advance of the imaging technology,endoscopic procedures can be used to accurately examine the mucosa thatcovers the gastrointestinal cavities, and to detect small and largepathological lesions, such as inflammatory tissue, polyps,pseudo-polyps, serrated lesions, adenomas, ulcerations, dysplasias,pre-neoplastic and neoplastic formations, tumors and similar. Inaddition, endoscopic procedures in the gastrointestinal tract allow thedoctor to perform minor, surgical or non-surgical interventions, whichcomprise, for example, biopsies and removal of pathologic lesions(polyps, adenomas, dysplasias, Barrett's dysplasia, pre-neoplastic andneoplastic formations, tumors). Surgical interventions include twoendoscopic resection procedures commonly used in gastrointestinalendoscopy to remove pathological lesions: endoscopic mucosal resection(EMR) and endoscopic submucosal dissection (ESD). These two techniqueshave provided new alternatives for minimally invasive treatment ofgastrointestinal polyps, adenomas, dysplasias, Barrett's dysplasia andearly-stage cancers that involve a minimum risk of lymph-nodemetastasis. EMR is an endoscopic technique developed for removal ofsessile or flat neoplasms confined to the superficial layers (mucosa andsubmucosa) of the GI tract. EMR is typically used for removal of lesionssmaller than 2 cm or piecemeal removal of larger lesions. EMR also playsan important role in the assessment of resected specimens for accuratepathological staging. In contrast to polypectomy, EMR involves thelifting up of a lesion from the muscular layer by injecting a fluidagent, commonly normal saline (NS) solution, into the submucosal layer.EMR is also useful for obtaining specimens for accuratehistopathological staging to determine the risk of lymph-nodemetastasis. EMR facilitates the complete removal of the affected mucosaby excising through the middle or deeper portion of the gut wallsubmucosa. Various EMR techniques have been described and four methodsinvolving snare resection are commonly used: (1) the inject and cutmethod; (2) the inject, lift, and cut method; (3) cap-assisted EMR(EMRC); and (4) EMR with ligation (EMRL). The inject and cut technique,also known as submucosal injection polypectomy, has become widely usedin recent years because of its simplicity. The diseased mucosa is liftedup from the muscular layer by creating a submucosal fluid cushion,captured, strangulated using an electrosurgical snare, and thenresected. However, injection into the thin submucosal layer is adelicate process, the injected solution tends to dissipate quickly, flatand depressed lesions are hard to capture with the snare compared withprotruded lesions, and large or awkwardly located lesions can bedifficult to remove (Uraoka et al., Drug Design, Development and Therapy2008:2 131-138). Injection-assisted EMR is frequently used for largeflat colon polyps.

Endoscopic submucosal dissection (ESD), a relatively new endoscopicresection procedure, was specifically developed for removing largerlesions. Lesions are dissected directly along the submucosal layer usingan electrosurgical knife, resulting in an en-bloc resection of evenlarge lesions. ESD has been predicted to replace conventional surgery intreating certain cancerous stages, but since it has a higher rate ofperforation and bleeding complications than conventional EMR, a greaterdegree of endoscopic skill and experience is required than for EMR.Various submucosal injection solutions had previously been developed andshown to be satisfactory for use during EMR, but introduction of thelengthier ESD procedure required a longer-lasting solution to helpidentifying the cutting line during dissection of the submucosal layer(Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138).

The use of submucosal injection is essential for a successful EMR, asinjection of fluid into the submucosa cushions facilitates the isolationof the tissue to be removed just before capture of the target lesionwith a snare, thereby reducing thermal injury and the risk ofperforation and haemorrhage while also facilitating an en-blocresection. Submucosal injection is considered to play an important rolein the EMR procedure, and the “ideal” submucosal injection solutionshould be both long-lasting as regards cushion duration and capable ofproducing a hemispheric shape to facilitate snaring. In addition,providing a sufficiently high submucosal elevation is important for safesubmucosal cutting during the ESD procedure (Uraoka et al., Drug Design,Development and Therapy 2008:2 131-138).

The ideal solution for injection-assisted EMR should be safe,inexpensive, non toxic, readily available, easy to inject and especiallyit should be capable of providing a high, long-lasting submucosalcushion. Wound healing characteristics should be also requested tofacilitate the closure of the wound created by the removal of theresected mucosa, as well as the presence of a colouring agent (such as adye) to allow an improvement in distinguishing more easily the deepnessof the muscolaris mucosa, avoiding undue perforation during ESD.

Normal saline solution (NS) has been commonly used for this purpose, butit is difficult to produce the proper submucosal fluid cushion andmaintain the desired height, particularly for flat elevated lesions,because of the rapid dispersion of the solution through the mucosallayers and absorption of NS into the surrounding tissue (Uraoka et al.,Drug Design, Development and Therapy 2008:2 131-138). For this reason,in long-lasting procedures and in the removal of large lesions, such aslarge flat polyps, repeated injection of the solution into thesubmucosal layer are required, with a consequent operationalcomplication for the personnel of the endoscopic unit.

In order to overcome the fast disappearance of the cushion, whichrepresents a typical problem encountered with NS, during the past decadeseveral types of solutions have been described and tested for the use insolution-assisted EMR. Each type of solution has its advantages anddisadvantages. For example, hyaluronic acid (HA) solutions have beenreported as the best agents for submucosal injections. HA solutionsprovide long-lasting fluid cushions and allow high successful en-blocresections and low perforation complication rates. Moreover, HA is safe,biocompatible and non-toxic, since it is a physiological component ofthe extracellular matrix. The main disadvantage of HA is its high cost,which renders it quite inaccessible for most endoscopic units. Othersolutions have been tested and described, such hypertonic dextrose andhydroxypropyl methylcellulose (HPMC), which however have been reportedto cause tissue damage and inflammation. Another recently investigatedinjection solution is fibrinogen mixture (FM) solution, which has a highviscosity and produces a long-lasting submucosal elevation, thuslowering the number of injections per lesion and shortening proceduretimes; in addition, FM is inexpensive. The main disadvantage of FM isthe possible the risk of transmission of viruses: since FM is obtainedby the fractionalization of coagulation proteins in human serum,contamination with hepatitis or other viruses is possible. As aboveillustrated, an ideal solution for EMR and ESD has not yet beendeveloped, and many researches in this field are still on-going.

Ideally, viscous solutions such as HA solutions or HPMC solutions couldmeet the requirements of the endoscopic resection procedures, since theycould provide a high and long-lasting cushion because of the lowtendency of the water coordinated by these polymers to diffuse andspread out in the tissues surrounding the lesion. However, the use ofviscous solutions, such as HA solutions or HPMC solutions, poses somechallenges in the procedure, due to the difficulty to get a viscoussolution flowed through the injection devices. As a matter of facts, ingastrointestinal EMR and ESD procedures, the injections of thecushion-forming solutions are performed using endoscopic injectionneedles. As well known in the art, endoscopic injection needles aredevices which can be long up to about 230 cm and which include arelatively long catheter within which an inner injection tube having adistal injection needle is slideably disposed. A proximal actuatinghandle is coupled to the catheter and the injection tube for moving onerelative to the other when necessary. Fluid access to the injection tubeis typically provided via a luer connector on the handle. Endoscopicinjection needle devices are typically delivered to the injection sitethrough the working channel of the endoscope. In order to protect thelumen of the endoscope working channel from damage, the handle of theinfusion needle device is manipulated to withdraw the distal injectionneedle into the lumen of the catheter before inserting the device intothe endoscope. This is important to prevent exposure of the sharp pointof the injection needle as the device is moved through the lumen of theendoscope. When the distal end of the endoscopic injection needle deviceis located at the injection site, its handle is again manipulated tomove the injection needle distally out of the lumen of the catheter.When advanced to the most distal position, the exposed portion of theinjection needle is approximately 4-6 mm in length. After the injectionsite has been pierced, the solution, usually contained in a 5 mL to 10mL syringe provided with a luer-lock fitting connected to the handle ofthe injection needle, is delivered through the injection tube and theneedle into the injection site.

The injection needle and other accessories commonly used duringendoscopic procedures, such as snares for polypectomy, clipping devices,biopsy forceps and similar, are passed through one or more specificchannels of the endoscope, usually called working channels or operatingchannels. Depending upon the type of endoscope used in GI endoscopy(e.g. gastroscope, enteroscope, colonoscope, duodenoscope, sigmoidoscopeand similar), the inner diameter of the working channels may varyconsiderably. However, the most common endoscopes used in GI endoscopyhave working channels with inner diameter in the range from about 2 mmto about 5 mm. Generally, the manufacturers of endoscopic accessoriesproduce accessories having outer diameters which allow them to fit allthe working channels. In particular, as regards the endoscopic injectionneedles, the outer diameter of catheter ranges from 1.9 mm to 2.3 mm;thus, considering that the inner injection tube is contained in theouter catheter, its internal diameter is usually 1 mm or less. Such asmall diameter of the injection tube causes a high dynamic resistance tothe flowing of the solution; this is more valid and important when aviscous solution is used. For this reason, the viscous solutions usedfor EMRs and ESDs often need to be diluted before their use to make thesolutions able to flow through the injection needle, with a loss oftheir characteristics of providing a high and long-lasting cushion.WO2011/103245 A1 describes a kit and a method for delivering ainjectable solution to a tissue treatment site, for use in ESD. Said kitincludes a housing having a chamber, a proximal portion and a distalportion. An injectable solution having a viscosity greater than about10000 cP is provided in the chamber. The kit also includes a plungermovably positionable within the proximal portion of the chamber, theplunger provides a seal at the proximal end portion. A pressure gauge isalso provided with the kit. A handle is connected to the housing and aplunger advancing member having a plunger handle is connected thereto.The plunger advancing member is provided separate from the housing andincludes a distal portion configured for operably connecting with theproximal portion of the housing. The kit also includes an inner shaftprovided separate from the housing and having a proximal end portionconfigured for operably connecting with the distal portion of thehousing for receiving the injectable solution there through and a distalend configured for insertion in to the tissue treatment site. As askilled in the art would recognize, such a device allows the physicianto apply a pressure much higher than using a normal syringe, thusallowing the high viscous solution, having a viscosity of 10000 cP orgreater, to flow into the injection tube. Furthermore, WO2011/103245 A1discloses that suitable materials for inclusion in the injectablesolution include methylcelluloses, such as carboxymethyl cellulose (CMC)and hydroxypropyl methylcellulose (HPMC), extracellular matrix proteins,elastin, collagen, gelatin, fibrin, agarose, and alginate or mixturesthereof. However, the use of such a “high-pressure” generating deviceduring the endoscopic examination is known for being not favourablyaccepted by the experts of the field, since it is cumbersome, additionalwork is required to put it in place, it is difficult to be operatedtherefore it represents a complication of the EMR and ESD procedures.

Another tentative to overcome these issues is described in WO2009/070793A1 which discloses the use of purified inverse thermosensitive polymersin EMR. As well known in the art, inverse thermosensitive polymers arepolymers which, upon dissolution in solvents (such as water) in aconcentration above the critical micellar concentration (CMC), have thetendency to form micelles. At concentrations higher than the criticalgelation concentration (CGC), these micelles can order into a lattice;the result is a solution which shows inverse characteristics ofviscosity, which means that said solution displays an increase of itsviscosity with the temperature. Eventually, when the temperature israised above the critical gelation temperature (CGT), a gel forms. Thegelation is due to physical entanglement and packing of the micellarstructures, and it is reversible, thus the gel turns back to a liquidform when temperature is lowered below the critical gelationtemperature. Polymers of this kind are well known in the art, andcomprise, for examples, poloxamers (commercialized by BASF under thebrand name of Kolliphor™) and poloxamines (commercialized by BASF underthe brand name of Tetronic™). Aqueous solutions of those polymers atconcentrations above CGC can be liquid at room temperature and can forma gel once heated up to body temperature (i.e. about 37° C.).WO2009/070793 A1 discloses the use of a composition comprising apurified inverse thermosensitive polymer in an endoscopic procedure forgastrointestinal mucosal resectioning. Said composition, calledLeGoo-endo™, is an aqueous solution of purified poloxamer 237; it isdisclosed that the rapid reversible liquid to gel transition achieved asa result its purified nature allows LeGoo-endo™ to be liquid at roomtemperature and to become a gel only as it emerges from the catheter atthe EMR site, once heated to body temperature. WO2009/070793 A1 teachesthat, in order to obtain said rapid liquid to gel transition, the use ofa purified poloxamer was needed, and that said purified poloxamer wasobtained by a purification process aimed to the obtainment of a purifiedpolymer characterized by a lower polydipersity of the molecular weight.Moreover, WO2009/070793 A1 discloses that it was necessary to develop amethod of injecting through a catheter into the intestine or stomach apurified inverse thermosensitive polymer solution that transitions to agel at body temperature. Among the challenges overcome was the fact thatbecause the catheter quickly reaches body temperature while residentinside the body, the purified inverse thermosensitive polymer could gelinside the catheter prior to reaching the desired site for EMR.WO2009/070793 A1 discloses that the delivery problems were solved with asystem comprising a high-pressure needle catheter connected to a syringefilled with purified inverse thermosensitive polymer solution, whereinsaid high-pressure needle catheter was contained within anadministration device (e.g., a syringe pump) that generated pressure onthe plunger of the syringe through a manual (e.g., screw), electrical orpressurized-gas mechanism. As a matter of facts, in the in vivo example,WO 2009/070793 A1 discloses that five EMR were performed in the colon of2 pigs with LeGoo-endo™ using a 23-gauge scletotherapy needle with a5-mL syringe and a balloon dilator gun; LeGoo-endo™ was kept on iceduring the intervention. Saline containing syringes were also kept onice to cool the catheter immediately before poloxamer injections. As aperson skilled in the art will recognize, the operating proceduredisclosed by WO2009/070793 A1 is quite complex, for the followingreasons: it requires that the purified inverse thermosensitive polymersolution is kept on ice during the intervention; it requires the use ofa particular, high-pressure needle catheter; it requires that,immediately before the injection of the purified inverse thermosensitivepolymer solution, the catheter is cooled by means of injections of coldnormal saline solution kept on ice; it requires an administration device(e.g., a syringe pump) that generates pressure on the plunger of thesyringe to administer the purified inverse thermosensitive polymersolution.

U.S. Pat. No. 7,909,809 teaches a method for performing aninterventional endoscopic procedure in the gastrointestinal tract suchas polypectomy, endoscopic mucosal resection (EMR) and endoscopicsubmucosal dissection (ESD), said method comprising the administrationto a human of a bulking or cushioning material that has characteristicsof phase transition from a low viscosity state (e.g. liquid phase) intoa high viscosity state (e.g. gel phase) in response to a predeterminedtemperature (e.g. body temperature).

As delineated above, an ideal composition for endoscopic mucosalresection (EMR) and endoscopic submucosal dissection (ESD) has not yetbeen developed. As reported above, compositions in form of solutioncontaining, for example, HA (hyaluronic acid) are known in the art: HA(hyaluronic acid) solutions are viscous and capable of providinglong-lasting submucosal cushions; moreover, they are safe and non toxic.However, they are known to be highly expensive.

Cellulose derivatives, such as HPMC and CMC, are cheap and theirsolutions are capable of providing long-lasting submucosal cushions;however, due to their viscosity, a particular device such a syringe pumpis required to make them flow into the injection needle, thus they areknown for being difficult and uncomfortable to be injected.

Inverse thermosensitive polymers, such as poloxamers and poloxamines,are cheap and their solutions, in view of their capability of gelling atbody temperature (i.e. about 37° C.), are capable of providinglong-lasting submucosal cushions; it is however known in the art that,to obtain the gelification of the solution at body temperature (i.e.about 37° C.), such polymers need to be contained in the solution in aconcentration equal to or above the critical gelation concentration(CGC), which is the concentration at which the transition of phase fromsolution to gel occurs upon heating at or above the critical gelationtemperature (CGT). Accordingly, such polymers are usually contained inthe known solutions in an amount equal to or above the critical gelationconcentration (CGC). Similar concentrations of these polymers howevercause several drawbacks, such as the gelification of the solutioncontaining thereof inside the injection needle. A complex procedure isperformed in order to avoid that the solution gelled inside theinjection needle, namely keeping the composition on ice, cooling theinjection needle with cold NS (normal saline solution) then using asyringe pump to administer them, with evident disadvantages for theendoscopist.

Therefore, there is the need to provide a composition for use inendoscopic procedure (particularly in EMR and ESD) able to be safe,inexpensive, non toxic, readily available, easy to inject and at thesame time capable of providing a high, long-lasting submucosal cushion.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The first cushion generated by the injection of the compositionaccording to example 1 into the sub-mucosal layer of an ex-vivo porcinestomach.

FIG. 2: The second cushion generated by the second injection of thecomposition according to Example 1 into the sub-mucosal layer of anex-vivo porcine stomach.

FIG. 3: The first cushion of FIG. 1 after cut immediately after theinjection, a viscous product with a good consistency is visible into thesub-mucosal layer.

FIG. 4: A specimen of the mucosa of the first cushion of FIG. 1 afterresection, wherein is visible that the product formed by the compositioninjected remains attached to the excised piece.

FIG. 5: The second cushion of FIG. 2 after 15 minutes from injection.

FIG. 6: The second cushion of FIG. 2 after cut, a viscous product with agood consistency similar to that of FIG. 3 is visible into thesub-mucosal layer.

FIG. 7: A specimen of the mucosa of the second cushion of FIG. 2 afterresection, wherein is visible that the product formed by the compositioninjected remains attached to the excised piece.

FIG. 8: The graph showing the microemulsion droplet size distribution byintensity, measured on the composition of Example 15 after step a) ofthe manufacturing process (see also example 9 and the Table A of Example17).

FIG. 9: The graph showing the microemulsion droplet size distribution byintensity, measured on the composition of example 15 after step d) ofthe manufacturing process (see also Example 9 and the Table B of Example17).

FIG. 10: Superimposing graph showing the comparison between themicroemulsion droplet size distribution by intensity after step a) andafter step d) of the manufacturing process, measured on the compositionof example 15 (see also Example 9 and the Table C of Example 17).

FIG. 11: The graph showing the microemulsion droplet size distributionby intensity (three replicates on the same sample), measured on thecomposition of Example 15 after step e) of the manufacturing process(see also Example 9 and the Table D of Example 17).

FIG. 12: Height of the sub-mucosa cushion formed upon injection of asuitable volume of the composition of Example 11, at time 0 and after 60minutes.

FIG. 13: The image shows an endoscopy in a minipig stomach (in vivo testin minipig of Example 21), and in particular the endoscopic injectionneedle, contained in the working channel of the endoscope, injecting thecomposition of Example 5 into the submucosal layer.

FIG. 14: The image shows an endoscopy in a minipig stomach (in vivo testin minipig of Example 21), and in particular the submucosal cushion atthe end of the administration. The interventional area has a bluecontrasting colour compared to the surrounding area.

FIG. 15: The image shows an endoscopy in the minipig stomach (in vivotest in minipig of Example 21) after 24 hours from the administration ofthe composition of Example 5. In the area where the composition wasinjected, the submucosal cushion is no longer visible. The gastricmucosa showed no gross macroscopic changes due to the administration ofthe composition.

FIG. 16: The graph showing the rheogram viscosity versus temperature.

SUMMARY OF THE INVENTION

The invention herein disclosed provides a pharmaceutical composition inform of emulsion or microemulsion and the use thereof in endoscopicprocedures, preferably gastrointestinal endoscopic procedures.

The invention provides a pharmaceutical composition in form of emulsionor microemulsion for use in an endoscopic procedure, wherein saidpharmaceutical composition comprises at least one inversethermosensitive polymer and optionally at least one physiologicallyacceptable excipient. Preferably, said endoscopic procedure comprisesthe administration of said pharmaceutical composition to a human.

The invention herein disclosed provides a method for performing anendoscopic procedure, wherein said pharmaceutical composition comprisesat least one inverse thermosensitive polymer and optionally at least onephysiologically acceptable excipient. Preferably, said method comprisesthe administration of a pharmaceutical composition in form of emulsionor microemulsion to a human.

DESCRIPTION OF THE INVENTION

The invention herein disclosed provides a pharmaceutical composition inform of emulsion or microemulsion wherein said pharmaceuticalcomposition comprises at least one inverse thermosensitive polymer in anamount below the critical gelation concentration (CGC) and wherein saidpharmaceutical composition remains in liquid phase up to a temperatureof about 40° C. in laboratory test conditions and the use thereof inendoscopic procedures. Preferably, said endoscopic procedures aregastrointestinal endoscopic procedures. More preferably, saidgastrointestinal endoscopic procedures are performed in the esophagus,stomach and/or small intestine (duodenum, jejunum, ileum), in the cecum,in the colon, in the sigmoid colon and/or in the rectum.

The invention herein disclosed provides a pharmaceutical composition inform of emulsion or microemulsion for use in endoscopic procedures,wherein said pharmaceutical composition comprises at least one inversethermosensitive polymer in an amount below the critical gelationconcentration (CGC) and wherein said pharmaceutical composition remainsin liquid phase up to a temperature of about 40° C. in laboratory testconditions.

The invention herein disclosed provides a method for performing anendoscopic procedure, said method comprising the administration of apharmaceutical composition in form of emulsion or microemulsion to ahuman, wherein said pharmaceutical composition comprises at least oneinverse thermosensitive polymer in an amount below the critical gelationconcentration (CGC) and wherein said pharmaceutical composition remainsin liquid phase up to a temperature of about 40° C. in laboratory testconditions.

According to the invention, said endoscopic procedure is preferably anendoscopic resection performed during a gastrointestinal endoscopy, morepreferably a polypectomy, an endoscopic mucosal resection (EMR) and/oran endoscopic submucosal dissection (ESD).

According to the invention, said gastrointestinal endoscopy ispreferably performed in the esophagus, stomach and/or small intestine(duodenum, jejunum, ileum), in the cecum, in the colon, in the sigmoidcolon and/or in the rectum.

Further, the invention herein disclosed provides a kit for use in anendoscopic procedure, said kit comprising a pharmaceutical compositionin form of emulsion or microemulsion, an endoscopic injection needle, asyringe and instruction for use thereof, wherein said pharmaceuticalcomposition in form of emulsion or microemulsion comprises at least oneinverse thermosensitive polymer and wherein said endoscopic procedure ispreferably an endoscopic resection of the mucosa performed during agastrointestinal endoscopy, more preferably a polypectomy, an endoscopicmucosal resection (EMR) and/or an endoscopic submucosal dissection(ESD).

More in details, the pharmaceutical composition in form of emulsion ormicroemulsion is injected in a target tissue in order to form a cushion,which may be then optionally subjected to an endoscopic surgicalprocedure, such as a resection procedure.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have been working to find out innovative pharmaceuticalcompositions for use in endoscopic procedures, preferably polypectomy,endoscopic mucosal resection (EMR) and/or endoscopic mucosal resection(ESD) which embodies the characteristics requested by endoscopicphysicians, especially safety, inexpensiveness, absence of toxiceffects, easiness to be injected and capacity of providing a high,long-lasting sub-mucosal cushion.

It was surprisingly discovered that pharmaceutical compositions in formof emulsions or microemulsions which comprise at least one inversethermosensitive polymer in an amount below the critical gelationconcentration (CGC) and remain in liquid phase up to a temperature ofabout 40° C. in laboratory test conditions, show the ability to form ahigh, long-lasting sub-mucosal cushion being meanwhile safe,inexpensive, non toxic and easy to be injected. It is therefore clearthe consequent improvement given by these compositions in endoscopicprocedures, particularly in the endoscopic resection duringgastrointestinal endoscopy.

The high, long-lasting submucosal cushion has been surprisingly observedby the inventors once the composition in form of emulsion ormicroemulsion according to the invention herein disclosed was injectedinto the submucosal layer of an ex vivo porcine stomach, whichconstitutes a well known model of the human gastrointestinal mucosa (SooHoon Eun et al. “Effectiveness of Sodium Alginate as a SubmucosalInjection Material for Endoscopic Mucosal Resection in Animal”, Gut andLiver, Vol. 1, No 1, June 2007, pp 27-32).

As well known in the art, inverse thermosensitive polymers are polymerswhich, upon dissolution in water in a concentration above the criticalgelation concentration (CGC), provide solutions that show inversecharacteristics of viscosity, which means that said solutions display anincrease of their viscosity with the temperature. In particular, aqueoussolutions of said polymers form gels above the critical gelationconcentration (CGC), when the temperature is raised above the criticalgelation temperature (CGT). The gelation is due to physical entanglementand packing of the micellar structures, and it is reversible, thus thegel turns back to a liquid form when temperature is lowered below thecritical gelation temperature. Polymers of this kind are well known inthe art, and comprise, for examples, poloxamers (commercialized by BASFunder the brand name of Kolliphor™) and poloxamines (commercialized byBASF under the brand name of Tetronic™). As well known in the art, eachkind of poloxamer has a characteristic critical gelation concentration(CGC); among the poloxamers, poloxamer 407 has the lowest CGC. Asreported in Evren Algin Yapar et al., Tropical Journal of PharmaceuticalResearch October 2012; 11 (5): 855-866, in order to attain relativelystable gels, these applications require polymer concentrations ofusually equal to or above 15% by weight, with respect to the weight ofthe solution. Moreover, J. J. Escobar-Chavez et al., Journal of Pharmacy& Pharmaceutical Sciences, 9(3):339-358, (2006) reports that poloxamer407 is of particular interest since concentrated solutions (>20% w/w) ofthe copolymer are transformed from low viscosity transparent solutionsto solid gels on heating to body temperature. As already mentioned,aqueous solutions of said polymers form gels above the critical gelationconcentration (CGC), when the temperature is raised above the criticalgelation temperature (CGT). The critical gelation temperature (CGT) canbe modulated by varying the concentration of the inverse thermosensitivepolymer, which means that the higher the concentration of said polymer,the lower the critical gelation temperature (CGT). As well known in theart, the gel-forming ability of solutions of inverse thermosensitivepolymers requires that the concentration of said polymers in saidsolutions is equal to or above the critical gelation concentration(CGC): if such polymers are contained in an amount below the CGC, thesolutions do not transition from a liquid phase to a gel phase inresponse to the raise in temperature. At the time the invention wasmade, it was thought in the art that the ability to form a gel uponheating to body temperature (i.e. about 37° C.) in laboratory testconditions characteristic of aqueous solutions containing some kinds ofinverse thermosensitive polymers in an amount equal to or above thecritical gelation concentration (CGC), was an essential feature forensuring the formation of a long-lasting submucosal cushion once saidsolutions were injected into the submucosal layer of thegastrointestinal tract. As a matter of facts, it was thought that saidsolutions were able to form a long-lasting submucosal cushion uponinjection into the submucosal layer of the gastrointestinal tract due tothe transition to a gel phase, in response to the raise in thetemperature (i.e. the body temperature). Thus, it was thought in the artthat the ability of aqueous solutions containing an inversethermosensitive polymer in an amount equal to or above the criticalgelation concentration (CGC) to form a long-lasting submucosal cushionupon injection into the submucosal layer of the gastrointestinal tractwas bound to their ability to gel upon heating at body temperature (i.e.about 37° C.) in laboratory test conditions. In other words, it wasthought that, in order to ensure the formation of a long-lasting cushioninto the submucosal layer of the gastrointestinal tract, said solutionshad to contain an inverse thermosensitive polymer at a concentrationequal to or above the critical gelation concentration (CGC), since onlyin this case said solutions would have been able to transition from aliquid phase to a gel phase in response to the raise in temperature(e.g. the body temperature).

It was now discovered that pharmaceutical compositions in form ofemulsions or microemulsions according to the invention herein disclosed,which do not have the ability to form a gel (i.e. remain in a liquidphase) up to a temperature of about 40° C. in laboratory testconditions, preferably upon heating at body temperature (i.e. about 37°C.), are surprisingly able to form a high, long-lasting submucosalcushion once injected into the submucosal layer of a porcine stomach(ex-vivo). In particular, in a comparison test foreseeing the injectionof different compositions into the submucosal layer of a porcine stomachmaintained at a temperature of about 37° C., it was surprisinglydiscovered that pharmaceutical compositions in form of emulsions ormicroemulsions according to the invention herein disclosed, even thoughunable to gel upon heating at body temperature (i.e. about 37° C.) inlaboratory test conditions, were surprisingly able to form a high,long-lasting cushion in the above submucosal layer (porcine stomachex-vivo) similar for height, shape and duration to that formed byconventional solutions, i.e. comprising an inverse thermosensitivepolymer at a concentration above the critical gelation concentrationwhich were able to gel upon heating at body temperature (i.e. about 37°C.) in laboratory conditions.

It was therefore surprisingly discovered that the absence of gellingproperties, observed in laboratory test conditions for thepharmaceutical compositions in form of emulsions or microemulsionsaccording to the invention herein disclosed, was not related to theability of forming a submucosal cushion of said pharmaceuticalcompositions observed in the porcine stomach (ex-vivo). As a personskilled in the art will recognize, such results were unexpected andunobvious as well as of significant advantage in the endoscopicprocedures. In the known art, it was in fact taught that the gel-formingability of solutions of inverse thermosensitive polymers upon heating atbody temperature (i.e. about 37° C.), in laboratory conditions, wasrelated to the gel-forming ability of said solutions in ex-vivo or inin-vivo models of gastrointestinal mucosal resectioning procedures.

The inventors have surprisingly found that the pharmaceuticalcompositions in form of emulsions or microemulsions according to theinvention herein disclosed have the ability of forming a submucosalcushion in ex vivo and/or in in vivo models of gastrointestinal mucosalresectioning procedures, even though the inverse thermosensitive polymerconcentration is contained in said pharmaceutical compositions in anamount below its critical gelation concentration (CGC) and,consequently, said pharmaceutical compositions are unable to gel up to atemperature of about 40° C., especially upon heating at body temperature(i.e. about 37° C.), in laboratory test conditions.

The invention herein disclosed provides a pharmaceutical composition inform of emulsion or microemulsion, wherein said pharmaceuticalcomposition comprises at least one inverse thermosensitive polymer in anamount below the critical gelation concentration (CGC) and wherein saidpharmaceutical composition remains in liquid phase up to a temperatureof about 40° C. in laboratory test conditions and the use thereof inendoscopic procedures.

The invention herein disclosed provides a pharmaceutical composition inform of emulsion or microemulsion for use in an endoscopic procedure,wherein said composition comprises at least one inverse thermosensitivepolymer in an amount below the critical gelation concentration (CGC),and wherein said composition remains in liquid phase up to a temperatureof about 40° C. in laboratory test conditions.

According to the invention, said endoscopic procedure comprises theadministration of said pharmaceutical composition to a human.

According to the invention said endoscopic procedure is preferably agastrointestinal endoscopic procedure, more preferably performed in theesophagus, stomach, small intestine (duodenum, jejunum, ileum), in thececum, in the colon, in the sigmoid colon and/or in the rectum.).

More in details, the pharmaceutical composition is injected in a targettissue of said human in order to form a cushion which is then optionallysubjected to an endoscopic surgical procedure, such as a resectionprocedure,

The invention herein disclosed thus provides also a method forperforming an endoscopic procedure, said method comprising theadministration of a pharmaceutical composition in form of emulsion ormicroemulsion to a human, wherein said composition comprises at leastone inverse thermosensitive polymer in an amount below the criticalgelation concentration (CGC), and wherein said composition remains inliquid phase up to a temperature of about 40° C. in laboratory testconditions. More in details, the pharmaceutical composition is injectedin a target tissue of said human in order to form a cushion which isthen optionally subjected to an endoscopic surgical procedure, such as aresection procedure.

According to the invention, the pharmaceutical composition in form ofemulsion or microemulsion preferably remains in liquid phase at atemperature comprised between about 5° C. and 40° C., more preferably atabout 5° C., about 20° C., about 25° C., about 30° C. and/or about 37°C. in laboratory test conditions.

According to a preferred embodiment of the invention, the pharmaceuticalcomposition in form of emulsion or microemulsion remains in liquid phaseboth at room temperature (i.e. about 20-25° C.) and at body temperature(i.e. about 37° C.) in laboratory test conditions.

According to another preferred embodiment, the pharmaceuticalcomposition in form of emulsion or microemulsion of the invention has aviscosity below about 150 cP (centipoises), more preferably below about100 cP (centipoises), much more preferably below about 50 cP(centipoises). According to another preferred embodiment thepharmaceutical composition in form of emulsion or microemulsion of theinvention has a viscosity below about 20 cP (centipoises), morepreferably below about 10 cP. According to the invention, said viscosityis preferably measured at about 25° C., at about 30° C. and/or at about37° C., more preferably using a Brookfield LVDV-III Ultra ProgrammableRheometer™ equipped with a Brookfield Small Sample Adapter™ device andusing a Brookfield™ spindle N. 31.

Alternatively, said viscosity is measured using a Brookfield LVDV-IIIUltra Programmable Rheometer™ equipped with a Brookfield Enhanced ULAdapter™ device and using a Brookfield™ spindle N. 00.

According to another preferred embodiment of the invention, saidendoscopic procedure is an endoscopic resection procedure performedduring a gastrointestinal endoscopy, preferably a polypectomy, anendoscopic mucosal resection (EMR) and/or an endoscopic submucosaldissection (ESD).

According to the invention said endoscopic procedure is preferably agastrointestinal endoscopic procedure, more preferably performed in theesophagus, stomach, small intestine (duodenum, jejunum, ileum), in thececum, in the colon, in the sigmoid colon and/or in the rectum.

According to the invention, said polypectomy, endoscopic mucosalresection (EMR) and/or said endoscopic submucosal dissection (ESD) areused for the removal of mucosal lesions, polyps, pseudo-polyps, flatpolyps, adenomas, serrated lesions, dysplasias, Barrett's dysplasia,pre-neoplastic formation, neoplastic formations and/or tumors duringgastrointestinal endoscopy.

According to the invention, said polypectomy, endoscopic mucosalresection (EMR) and/or said endoscopic submucosal dissection (ESD) arealso used for the removal of pathologic and/or dysplastic mucosal tissuein case of esophagitis, erosive esophagitis, Barrett's esophagus (suchas in ablation procedures), and/or gastrointestinal pathologicalhypersecretory conditions, such as Zollinger Ellison Syndrome.

According to an embodiment, said pharmaceutical composition in form ofemulsion or microemulsion is administered to a human through injectionby means of an endoscopic injection needle provided with a retractableneedle and of a syringe.

According to the invention, said pharmaceutical composition in form ofemulsion or microemulsion can be preferably a water-in-oil emulsion ormicroemulsion, or an oil-in-water emulsion or microemulsion. Accordingto a preferred embodiment, the pharmaceutical composition in form ofemulsion or microemulsion is an oil-in-water emulsion or microemulsion.

According to an embodiment, said pharmaceutical composition in form ofemulsion or microemulsion for use in an endoscopic procedure comprises:

-   -   (a) an aqueous phase;    -   (b) an oily phase;    -   (c) at least one surfactant;    -   (d) at least one inverse thermosensitive polymer,    -   (e) optionally at least one physiologically acceptable        excipient;        wherein said at least one inverse thermosensitive polymer is        comprised in an amount below the critical gelation concentration        (CGC), and wherein said composition is in liquid phase up to a        temperature of about 40° C. in laboratory test conditions.

According to another embodiment, said pharmaceutical composition in formof emulsion or microemulsion for use in an endoscopic procedurecomprises:

-   -   (a) an aqueous phase;    -   (b) an oily phase;    -   (c) at least one surfactant;    -   (d) at least one inverse thermosensitive polymer;    -   (e) optionally at least one co-surfactant;    -   (f) optionally at least one physiologically acceptable        excipient;        wherein said at least one inverse thermosensitive polymer is        comprised in an amount below the critical gelation concentration        (CGC), and wherein said composition is in liquid phase up to a        temperature of about 40° C. in laboratory test conditions.

According to another embodiment, said pharmaceutical composition in formof emulsion or microemulsion for use in an endoscopic procedurecomprises:

-   -   (a) an aqueous phase;    -   (b) an oily phase;    -   (c) at least one surfactant;    -   (d) at least one inverse thermosensitive polymer;    -   (e) optionally at least one co-surfactant;    -   (f) at least one dye;    -   (g) optionally at least one physiologically acceptable        excipient;        wherein said at least one inverse thermosensitive polymer is        comprised in an amount below the critical gelation concentration        (CGC), and wherein said composition is in liquid phase up to a        temperature of about 40° C. in laboratory test conditions.

According to another embodiment, said pharmaceutical composition in formof emulsion or microemulsion for use in an endoscopic procedurecomprises:

-   -   (a) an aqueous phase;    -   (b) an oily phase;    -   (c) at least one surfactant;    -   (d) at least one inverse thermosensitive polymer;    -   (e) optionally at least one co-surfactant;    -   (f) at least one dye;    -   (g) optionally at least one agent characterized by having a        trophic activity on the epithelial cells of the gastrointestinal        mucosa;    -   (h) optionally at least one physiologically acceptable        excipient;        wherein said at least one inverse thermosensitive polymer is        comprised in an amount below the critical gelation concentration        (CGC), and wherein said composition is in liquid phase up to a        temperature of about 40° C. in laboratory test conditions.

According to another embodiment, said pharmaceutical composition in formof emulsion or microemulsion for use in an endoscopic procedurecomprises:

-   -   (a) an aqueous phase;    -   (b) an oily phase;    -   (c) at least one surfactant;    -   (d) at least one inverse thermosensitive polymer;    -   (e) optionally at least one co-surfactant;    -   (f) at least one dye;    -   (g) optionally at least one agent characterized by having a        trophic activity on the epithelial cells of the gastrointestinal        mucosa;    -   (h) optionally at least one therapeutic agent;    -   (i) optionally at least one physiologically acceptable        excipient;        wherein said at least one inverse thermosensitive polymer is        comprised in an amount below the critical gelation concentration        (CGC), and wherein said composition is in liquid phase up to a        temperature of about 40° C. in laboratory test conditions.

According to another embodiment, the present invention relates to apharmaceutical composition in form of emulsion or microemulsion whichcomprises, consists or essentially consists of:

-   -   (a) an aqueous phase;    -   (b) an oily phase;    -   (c) at least one surfactant;    -   (d) at least one inverse thermosensitive polymer,    -   (e) optionally at least one co-surfactant;    -   (f) optionally at least one dye;    -   (g) optionally at least one agent characterized by having a        trophic activity on the epithelial cells of the gastrointestinal        mucosa;    -   (h) optionally at least one therapeutic agent;    -   (i) optionally at least one physiologically acceptable        excipient;        wherein said at least one inverse thermosensitive polymer is        comprised in an amount below the critical gelation concentration        (CGC), and wherein said composition is in liquid phase up to a        temperature of about 40° C. in laboratory test conditions.

According to the invention, the pharmaceutical composition in form ofemulsion or microemulsion preferably remains in liquid phase at atemperature comprised between about 5° C. and about 40° C., morepreferably at about 5° C. and/or about 20° C. and/or about 25° C. and/orabout 30° C. and/or about 37° C., in laboratory test conditions.

According to the invention herein disclosed, the main component of theaqueous phase of said pharmaceutical composition is water for injection.As well known in the art, water for injection represents a highlypurified, distilled water, free of salts and of carbon contaminants, andfree of microorganisms and of bacterial endotoxines. Water for injectionis water purified by distillation or a purification process that isequivalent or superior to distillation in the removal of chemicals andof microorganisms. In some embodiments of the invention hereindisclosed, said aqueous phase may comprise, in dissolved form, one ormore inorganic salts selected form the group comprising, but not limitedto: chlorides, bromides, iodides, phosphates, carbonates, bicarbonates,sulfates, nitrates and the like. In some embodiments, said aqueous phasemay comprise, in dissolved form, one or more organic salts selected formthe group comprising, but not limited to: citrates, maleates, fumarates,acetates, lactates and the like. Any mixture of the above inorganic andorganic salts may be used to form the appropriate pharmaceuticalcomposition, generally to buffer the pH of the composition in suitablebiocompatible ranges or to reach the osmotic pressure required by thebiologic environment where said pharmaceutical composition has to beinjected. In some embodiments, the aqueous phase of the pharmaceuticalcomposition herein disclosed may comprise an amount of one or moreinorganic and/or organic salts or mixtures thereof such as to have afinal pharmaceutical composition which is hypotonic. In someembodiments, the aqueous phase of the pharmaceutical composition hereindisclosed may comprise an amount of one or more inorganic and/or organicsalts or mixtures thereof such as to have a final pharmaceuticalcomposition which is isotonic. In some embodiments, the aqueous phase ofthe pharmaceutical composition herein disclosed may comprise an amountof one or more inorganic and/or organic salts or mixtures thereof suchas to have a final pharmaceutical composition which is hypertonic.According to the invention herein disclosed, said inorganic and/ororganic salts or mixtures thereof may be present in an amount rangingfrom 0% to 5% by weight with respect to the weight of the composition,more preferably from 0.1% to 4% by weight with respect to the weight ofthe composition, much more preferably from 0.5% to 3% by weight withrespect to the weight of the composition. According to a preferredembodiment, said inorganic and/or organic salts or mixtures thereof maybe present in an amount ranging from 0.3% to 0.7% by weight respect tothe weight of the composition.

In a preferred embodiment, the aqueous phase of said pharmaceuticalcomposition contains sodium chloride dissolved. According to the latterembodiment, said sodium chloride is present in an amount ranging fromabout 0% to about 5% by weight with respect to the weight of thecomposition, more preferably from about 0.1% to about 3% by weight withrespect to the weight of the composition, much more preferably fromabout 0.5% to about 2% by weight with respect to the weight of thecomposition.

According to a preferred embodiment, said sodium chloride may be presentin an amount ranging from 0.3% to 0.7% by weight respect to the weightof the composition. More preferably, said sodium chloride is present inan amount of about 0.44% w/w.

In some embodiments, the aqueous phase of the pharmaceutical compositionherein disclosed comprises a buffer. In some embodiments, said buffer isa phosphate buffer. In some embodiments, said buffer is a citratebuffer. In some embodiments, said buffer is a bicarbonate buffer. In apreferred embodiment, said buffer is a phosphate buffer added with oneor more inorganic salts unable to buffer the pH. According to the latterembodiment, the concentration of said phosphate buffer and saidinorganic salts unable to buffer the pH is such as to have an aqueousphase which is phosphate buffered saline (PBS). As well known in theart, PBS is a water-based salt solution containing sodium chloride,sodium phosphate, and, optionally, potassium chloride and potassiumphosphate; PBS for medical applications is an isotonic solution, i.e.its osmolarity and its pH match those of the human body. Severalcompositions and preparation methods of PBS are well known in the art.

According to the invention herein disclosed, the pH value of thepharmaceutical composition in form of emulsion or microemulsion rangesfrom about 4.0 to about 9.0, more preferably from about 5.0 to about8.0, much more preferably from about 5.5 to about 7.5. According to theinvention, the pH value of said pharmaceutical composition in form ofemulsion or microemulsion may be adjusted within the desired range bycommon techniques well known in the art, such as, for example, theaddition of physiologically acceptable bases and/or acids.

According to the invention herein disclosed, said oily phase of saidpharmaceutical composition comprises at least one lipophilic compound.In some embodiments, said at least one lipophilic compound may beselected in the group of natural oils, comprising, but not limited to:almond oil, canola oil, castor oil, corn oil, cottonseed oil, olive oil,safflower oil, sesame oil, soybean oil and the like. In someembodiments, said at least one lipophilic compound may be selected inthe group of fatty acid esters, comprising, but not limited to:isopropyl palmitate, isopropyl myristate, ethyl oleate and the like. Insome embodiments, said at least one lipophilic compound may be selectedin the group of fatty alcohols, comprising, but not limited to: myristicalcohol, oleyl alcohol and the like. In some embodiments, said at leastone lipophilic compound may be selected in the group of fatty acids,comprising, but not limited to: myristic acid, oleyl acid, palmitic acidand the like. In some embodiments, said at least one lipophilic compoundmay be selected in the group of triglycerides, such as long and/ormedium-chain triglycerides and the like. In some embodiments, said atleast one lipophilic compound may be selected in the group ofdiglycerides. In some embodiments, said at least one lipophilic compoundmay be selected in the group of monoglycerides. Any mixture of the abovelipophilic compounds can be used to form the appropriate pharmaceuticalcomposition. In one embodiment, the lipophilic compound of said oilyphase is sesame oil. In another embodiment, the lipophilic compound ofsaid oily phase is almond oil. In another embodiment, the lipophiliccompounds of said oily phase are medium-chain triglycerides. In apreferred embodiment, the lipophilic compound of said oily phase issoybean oil.

According to the invention herein disclosed, the oily phase of saidpharmaceutical composition ranges from about 0.001% to about 20% byweight with respect to the weight of the composition, preferably fromabout 0.01% to about 2% by weight with respect to the weight of thecomposition, more preferably from about 0.02% to about 1% by weight ofthe with respect to the weight of the composition. According to apreferred embodiment, said oily phase is contained in the composition ofthe invention in an amount from about 0.01% by weight to about 0.5% byweight, with respect to the weight of the composition.

More preferably, the oily phase is contained in the composition of theinvention in an amount of about 0.08% by weight or about 0.16% byweight, with respect to the weight of the composition. Much morepreferably, said oily phase is contained in the composition of theinvention in an amount of about 0.02% w/w or about 0.05% w/w or about0.1% by weight, with respect to the weight of the composition. Accordingto the invention herein disclosed, the pharmaceutical composition inform of emulsion or microemulsion contains at least one inversethermosensitive polymer at a concentration below the critical gelationconcentration (CGC). Accordingly, said pharmaceutical composition inform of emulsion or microemulsion is not able to transition from aliquid phase to a gel phase in response to the raise in temperature upto 40° C. in laboratory test conditions, such as from room temperature(i.e. about 20-25° C.) to body temperature (i.e. about 37° C.). Thus,said pharmaceutical composition in form of emulsion or microemulsionaccording to the invention herein disclosed is in liquid phase up to atemperature of about 40° C., preferably both at room temperature (i.e.about 20-25° C.) and at body temperature (i.e. about 37° C.) inlaboratory test conditions. Each type of inverse thermosensitive polymeris characterized by a specific critical gelation concentration (CGC);such concentrations are well known in the art and can be easily found inscientific literature. According to the invention herein disclosed, saidat least one inverse thermosensitive polymer may be selected in thegroup comprising, but not limited to: polyoxyethylene-polyoxypropyleneblock copolymers, such as poloxamers and the like. Said poloxamer may beselected in the group comprising, but not limited to: poloxamer 124,poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407 and the like.Any mixture of the above inverse thermosensitive polymers can be used toform the appropriate pharmaceutical composition. In a preferredembodiment, said at least one inverse thermosensitive polymer of saidpharmaceutical composition is poloxamer 188. In a preferred embodiment,said at least one inverse thermosensitive polymer of said pharmaceuticalcomposition is poloxamer 407. Further in another preferred embodiment,said at least one inverse thermosensitive polymer is a mixture ofpoloxamer 188 and poloxamer 407.

According to the invention, useful inverse thermosensitive polymers arebought on the market and used without any purification step.

According to the invention herein disclosed, said at least one inversethermosensitive polymer is present in an amount below the criticalgelation temperature (CGC), preferably below about 15% by weight withrespect to the weight of the pharmaceutical composition, more preferablybetween about 2% and about 14.5% by weight with respect to the weight ofthe pharmaceutical composition, much more preferably between about 3%and about 12% by weight with respect to the weight of the pharmaceuticalcomposition, further much more preferably between about 5% and about 11%by weight with respect to the weight of the pharmaceutical composition.Preferably, said at least one inverse thermosensitive polymer is presentin an amount of about 7%, or about 8%, or about 9%, or about 10% byweight with respect to the weight of the composition.

According to an embodiment, said at least one inverse thermosensitivepolymer is poloxamer 407 and it is contained in an amount of about 9% byweight with respect to the weight of the composition.

According to another embodiment, said at least one inversethermosensitive polymer is poloxamer 188 and it is contained in anamount of about 10% by weight with respect to the weight of thecomposition.

According to a further preferred embodiment, said at least one inversethermosensitive polymer is a mixture of poloxamer 188 and poloxamer 407,and such a mixture is contained in an amount of about 10% by weight withrespect to the weight of the composition.

According to the invention herein disclosed, said at least onesurfactant may be selected in the group of the non-ionic surfactants,comprising, but not limited to: PEG-fatty acid monoesters surfactants,such as PEG-15 hydroxystearate, PEG-30 stearate, PEG-40 laurate, PEG-40oleate and the like; PEG-fatty acid diesters surfactants, such as PEG-32dioleate, PEG-400 dioleate and the like; polyoxyethylene sorbitan fattyacid esters, such as polysorbate 20, polysorbate 60, polysorbate 80 andthe like; polyoxyethylene alkyl ethers, such as PEG-20 cetostearylether, polyoxyl 25 cetostearyl, cetomacrogol 1000 and the like; sorbitanfatty acid esters surfactants, such as sorbitan monolaurate, sorbitanmonopalmitate, sorbitan monooleate, and the like; propylene glycolesters of fatty acids; polyglycerol esters of fatty acids;polyoxyethylene castor oil derivatives such as polyoxyl 5 castor oil,polyoxyl 15 castor oil, polyoxyl 35 castor oil, polyoxyl 40 hydrogenatedcastor oil and the like; caprylocapryl polyoxyil-8 glycerides;polyoxylglycerides such as caprylocaproyl polyoxylglycerides, lauroylpolyoxylglycerides, oleoyl polyoxylglycerides and the like ceteareth 16,ceteareth 20, stearaeth 10, steareth 20, ceteth 20 and the like. Anymixture of the above non-ionic surfactant can be used to form theappropriate pharmaceutical composition. In one embodiment, the non-ionicsurfactant is polysorbate 80. In a preferred embodiment, the non-ionicsurfactant is PEG-15 hydroxystearate (also known aspolyoxyl-15-hydroxystearate).

According to the invention herein disclosed, said at least onesurfactant may be selected in the group of the ionic surfactants,comprising, but not limited to: egg lecithin, hydrogenated phosphatidylcholine from egg lecithin, soybean lecithin, hydrogenated soybeanlecithin, glycerophosphocholine, soybean lysolecithin, phospholipids,hydrogenated phospholipids, sodium lauryl sulphate and the like. Anymixture of the above ionic surfactant can be used to form theappropriate pharmaceutical composition. The above ionic surfactants arecommercialized by Lipoid, under the brand-name of Lipoid®. In oneembodiment, the ionic surfactant is egg lecithin. In another embodiment,the ionic surfactant is hydrogenated phosphatidyl choline from egglecithin. In another embodiment, the ionic surfactant is soybeanlecithin. Further in another embodiment, the ionic surfactant ishydrogenated soybean lecithin.

According to the invention herein disclosed, said at least onesurfactant is contained in an amount which ranges from about 0.001% toabout 10% by weight with respect to the weight of the pharmaceuticalcomposition, preferably from about 0.005% to about 5% by weight withrespect to the weight of the pharmaceutical composition, more preferablyfrom about 0.01% to about 2% by weight with respect to the weight of thepharmaceutical composition. According to a preferred embodiment, said atleast one surfactant is contained in an amount of about 0.08% or about0.1% or about 0.5% or about 0.6%, by weight with respect to the weightof the pharmaceutical composition.

The pharmaceutical composition of the invention herein disclosed maycomprise at least one co-surfactant. The addition of at least oneco-surfactant to the mixture oily phase-surfactant-aqueous phase isadvantageous since the co-surfactant acts in synergy with the surfactantin lowering the interfacial tension of the droplets of the dispersedphase of the emulsion or microemulsion, with a stabilizing effect on thesystem. In the preparation of pharmaceutical compositions in form ofemulsions or microemulsions according to the invention herein disclosed,said at least one co-surfactant can be selected in the groupscomprising, but not limited to: low and medium chain alcohols, such asethanol, propanol, isopropanol and the like; glycols, such as propyleneglycol and the like; polyethylene glycols, such as PEG 200, PEG 300, PEG400 and the like; DMSO; long chain alcohols, such as cetyl alcohol,myristyl alcohol, oleyl alcohol and the like; glycerol; low chainesters, such as ethyl acetate, ethyl lactate and the like; fatty acidesters, such as ethyl oleate, isopropyl myristate, isopropyl palmitateand the like; fatty acids, such as oleic acid, myristic acid and thelike; salts of fatty acids, such as sodium oleate, sodium palmitate,sodium stearate and the like. Any mixture of the above co-surfactantscan be used to form the appropriate pharmaceutical composition. In oneembodiment, the co-surfactant is propylene glycol. In anotherembodiment, the co-surfactant is glycerol. In another embodiment, theco-surfactant is sodium oleate. In a preferred embodiment, theco-surfactant is a mixture of glycerol and sodium oleate.

According to the invention herein disclosed, said at least oneco-surfactant is contained in an amount which ranges from about 0.00001%to about 1% by weight with respect to the weight of the pharmaceuticalcomposition, preferably from about 0.00005% to about 0.05% by weightwith respect to the weight of the pharmaceutical composition, morepreferably from about 0.0001% to about 0.01% by weight with respect tothe weight of the pharmaceutical composition.

The pharmaceutical composition of the invention herein disclosed maycomprise at least one dye. Dyes are widely used in compositions forendoscopic procedures. In particular, in compositions for EMR and/or ESDprocedures, the dyes are useful to feature the margins of the lesion tobe resected and the physiological structures underlying the mucosa;thus, the endoscopist can easily visualize the lesion he has to removeand he can perform the procedure with less risks of damaging thesubmucosal layer or the muscular tissue. The dye has the function torender immediately visible to the endoscopist the submucosal layer, sothat the surgical procedure is safer and there is a lower risk ofdamaging the structures beneath the mucosa, such as the submucosal layeritself and the external muscular wall.

In the preparation of the pharmaceutical composition according to theinvention herein disclosed, said at least one dye may be selected amongvital dyes (or absorptive dyes), non-vital dyes (or contrast dyes), andreactive dyes. Vital (or absorptive) dyes, such as Lugol's solution andmethylene blue, identify specific epithelial cell types by preferentialabsorption or diffusion across the cell membrane; non-vital (orcontrast) dyes, such as indigo carmine, seep through mucosal crevicesand highlight surface topography and mucosal irregularities; reactivedyes, such as congo red and phenol red, undergo chemical reactions withspecific cellular constituents, resulting in a colour change akin to apH indicator. According to the invention herein disclosed, said vital(or absorptive) dye may be selected in the group comprising, but notlimited to: Lugol's solution, methylene blue, toluidine blue, crystalviolet and the like. According to the invention herein disclosed, saidnon-vital (or contrast) dye may be selected in the group comprising, butnot limited to: indigo carmine and the like. According to the inventionherein disclosed, said reactive dye may be selected in the groupcomprising, but not limited to: Congo red, phenol red and the like. Anymixture of the above dyes can be used to form the appropriatepharmaceutical composition. According to a preferred embodiment, said atleast one dye is methylene blue. According to another preferredembodiment, said at least one dye is indigo carmine.

According to the invention herein disclosed, said at least one dye iscontained in an amount which ranges from about 0.0001% to about 0.2% byweight with respect to the weight of the pharmaceutical composition,preferably from about 0.0002% to about 0.05% by weight with respect tothe weight of the pharmaceutical composition, more preferably from about0.0005% to about 0.01% by weight with respect to the weight of thepharmaceutical composition. Much more preferably, said at least one dyeis contained in the composition of the invention in an amount of about0.001% by weight or about 0.002% by weight, with respect to the weightof the composition.

The pharmaceutical composition of the invention herein disclosed maycomprise at least one agent characterized by having a trophic activityon the epithelial cells of the gastrointestinal mucosa. Trophic agentsare substances capable of promoting cellular growth, differentiation,and survival. In gastrointestinal endoscopy, resectioning proceduressuch as polypectomy, EMR and/or ESD are generally not followed bysuturing. In other words, once the lesion has been removed by means ofone of said procedures, the mucosa is not sutured and the wound is leftopened; thus the healing of the wound must occur naturally. In thissense, the incorporation into the pharmaceutical compositions accordingto the invention of at least one agent proved to possess a trophicactivity on the epithelial cells of the gastrointestinal mucosa could beadvantageous, since said pharmaceutical compositions could exert apositive, beneficial effect on wound healing, promoting cellular growthand differentiation for a faster closure and healing of the surgicalwound.

In the preparation of pharmaceutical compositions in form of emulsionsor microemulsions according to the invention herein disclosed, said atleast one agent characterized by having a trophic activity on theepithelial cells of the gastrointestinal mucosa can be selected in thegroups comprising, but not limited to: aminoacids and salts thereof,such as arginine, glutamine, glutamic acid, citrulline, proline,cysteine and the like; short-chain fatty acids (SCFA) and salts thereof,such as acetic acid and salts thereof, propanoic acid and salts thereof,butyric acid and salts thereof, and the like; carbohydrates, such asglucose, fructose, galactose, sucrose, maltose, lactose and the like;polyamines and salts thereof, such as putresceine, spermidine, spermineand the like; fatty acids and salts thereof, such as oleic acid andsalts thereof, linoleic acid and salts thereof, mirystic acid and saltsthereof, stearic acid and salts thereof and the like; vitamins, such asvitamin A, vitamin B₂, vitamin C, vitamin D, and the like. Any mixtureof the above agents characterized by having a trophic activity on theepithelial cells of the gastrointestinal mucosa can be used to form theappropriate pharmaceutical composition. In one embodiment, the at leastone agent characterized by having a trophic activity on the epithelialcells of the gastrointestinal mucosa is sodium butyrate. In anotherembodiment, the at least one agent characterized by having a trophicactivity on the epithelial cells of the gastrointestinal mucosa issodium vitamin B₂. In a preferred embodiment, the at least one agentcharacterized by having a trophic activity on the epithelial cells ofthe gastrointestinal mucosa is glutamic acid.

According to the invention herein disclosed, said at least one agentcharacterized by having a trophic activity on the epithelial cells ofthe gastrointestinal mucosa is contained in an amount which ranges fromabout 0.01% to about 5% by weight with respect to the weight of thepharmaceutical composition, preferably from about 0.05% to about 3% byweight with respect to the weight of the pharmaceutical composition,more preferably from about 0.1% to about 2% by weight with respect tothe weight of the pharmaceutical composition.

The pharmaceutical composition of the invention herein disclosed maycomprise at least one therapeutic agent. In the preparation ofpharmaceutical compositions in form of emulsions or microemulsionsaccording to the invention herein disclosed, said at least onetherapeutic agent can be selected in the groups comprising, but notlimited to: antibiotics, such as penicillins, cephalosporins,aminoglycosides, macrolides, rifamycins, metronidazole and the like;non-steroidal anti-inflammatory drugs, such as ketorolac and saltsthereof, indometacin, piroxicam, ketoprofen and salts thereof, andmetamizol and salts thereof, and the like; steroidal anti-inflammatorydrugs, such as cortisol, prednisolone and esters thereof,methyprednisolone and esters thereof, triamcinolone acetonide,betamethasone and esters thereof, and the like; local anesthetics, suchas lidocaine and salts thereof, mepivacaine and salts thereof,bupuvacaine and salts thereof, and the like; vasoconstrictor drugs, suchas epinephrine and salts thereof, norepinephrine and salts thereof, andthe like. Any mixture of the above therapeutic agents can be used toform the appropriate pharmaceutical composition and to achieve specifictherapeutic effects. In an embodiment, said at least one therapeuticagent is a local anesthetic, such as lidocaine hydrochloride. In anotherembodiment, said at least one therapeutic agent is a vasoconstrictordrug, such as epinephrine hydrochloride. Further in another embodiment,the pharmaceutical composition according to the invention hereindisclosed comprises a local anaesthetic and a vasoconstrictor drug, suchas lidocaine hydrochloride and epinephrine hydrochloride.

Additionally, at least one physiologically acceptable excipient may beadded to the pharmaceutical composition according to the inventionherein disclosed to obtain final composition for use in endoscopicprocedures provided with suitable characteristics and stability. By wayof example, said at least one physiologically acceptable excipient maybe selected among antioxidants, chelating agents, preservatives,antimicrobial agents, polymers provided with bioadhesive properties,viscosity increasing agents, solvents and the like.

The pharmaceutical composition in form of emulsion or microemulsion ofthe invention herein disclosed may be packaged in primary packagingconfigurations well known in the art. Suitable primary packaging typescan be selected in the groups comprising, but not limited to: ampoules,vials, bottles, pre-filled syringes and the like. In an embodiment, thepharmaceutical composition in form of emulsion or microemulsion of theinvention herein disclosed is packaged in 5 mL or 10 mL pre-filledsyringes. In a preferred embodiment, the pharmaceutical composition inform of emulsion or microemulsion of the invention herein disclosed ispackaged in 10 mL, 20 mL or 50 mL vials. In another preferredembodiment, the pharmaceutical composition in form of emulsion ormicroemulsion of the invention herein disclosed is packaged in 10 mL, 20mL or 50 mL ampoules. The pharmaceutical composition in form of emulsionor microemulsion of the invention herein disclosed is administered bymeans of endoscopic injection needles. Preferably, the composition ismanually administered at room temperature.

Another aspect of the invention herein disclosed provides a kit for usein an endoscopic procedure, said kit comprising:

-   -   a) pharmaceutical composition in form of emulsion or        microemulsion according to the invention herein disclosed;    -   b) an endoscopic injection needle;    -   c) instruction for use.

In the preparation of said kit, said pharmaceutical composition in formof emulsion or microemulsion according to the invention herein disclosedmay be packaged in primary packaging configurations well known in theart. Suitable primary packaging types can be selected in the groupscomprising, but not limited to: ampoules, vials, bottles, pre-filledsyringes and the like. In an embodiment, in the preparation of said kit,the pharmaceutical composition in form of emulsion or microemulsion ofthe invention herein disclosed is packaged in 5 mL or 10 mL pre-filledsyringes. In a preferred embodiment, in the preparation of said kit, thepharmaceutical composition in form of emulsion or microemulsion of theinvention herein disclosed is packaged in 10 mL, 20 mL or 50 mL vials.In another preferred embodiment, in the preparation of said kit, thepharmaceutical composition in form of emulsion or microemulsion of theinvention herein disclosed is packaged in 10 mL, 20 mL or 50 mLampoules. In the preparation of said kit according to the inventionherein disclosed, suitable endoscopic injection needles may have adiameter of the needle ranging from 12 gauge to 35 gauge, preferablyfrom 15 gauge to 30 gauge, more preferably from 17 gauge to 28 gauge. Inthe preparation of said kit according to the invention herein disclosed,suitable endoscopic injection needles may have a length ranging from 100cm to 300 cm, preferably from 120 cm to 260 cm, more preferably from 140cm to 250 cm. In the preparation of said kit according to the inventionherein disclosed, suitable endoscopic injection needles may have anouter diameter ranging from 1.0 mm to 4.0 mm, preferably from 1.5 mm to3.0 mm, more preferably from 1.8 mm to 2.5 mm. In the preparation ofsaid kit according to the invention herein disclosed, suitableendoscopic injection needles may be composed of materials selected inthe groups comprising, but not limited to: polymers or copolymers, suchas polyethylene (PE), polypropylene (PP), polyvinylchloride (PVC),polycarbonate (PC), polytetrafluoroethylene (PTFE), polyethyleneterephthalate (PET), polystyrene (PS), polyamide (PA), epoxy resins,polyurethane, polyester, polymethyl methacrylate and the like; rubbers,such as silicone rubber, natural rubber and the like; metals and metalalloys such as aluminium, titanium, iron, chromium, nickel, molybdenum,stainless steel, and the like. Any combination of the above materialsmay be used to form the appropriate endoscopic injection needle.Endoscopic injection needles suitable for the preparation of the kitaccording to the invention herein disclosed can be found easily on themarket; by way of example, a suitable injection needle can be selectedfrom the marketed injection needles comprising, in a non-limiting wayCook® AcuJect® Variable Injection Needles, Cook® Injectaflow® VariableInjection Needles, Boston Scientific® Interject® Injection TherapyNeedles Catheters, G-Flex® Injection Needles, Endo-Flex® SclerotherapyNeedles, ConMed™ Click-Tip™ Injection Needles, Medi-Globe® Injectra®Injection Needle, Olympus®InjectorForce Max™, US Endoscopy™ Articulator™injection needle, US Endoscopy™ Vari-Safe™ injection needle,Kimberly-Clarck™ injection needle catheters, and the like.

In a preferred application of the invention, the pharmaceuticalcomposition in form of emulsion or microemulsion according to theinvention herein disclosed is used in an endoscopic resection procedureby sucking a volume of emulsion from its primary container by means of asyringe, injecting a suitable volume of said emulsion by means of anendoscopic injection needle inserted in the working channel of theendoscope immediately under the superficial mucosal layer, to depose aliquid volume into the submucosal layer that becomes a cushion when inplace: the elevation of the mucosal surface allow the endoscopist toperform an easy resection of the mucosal lesion found during theexecution of the endoscopic procedure even if the lesion is flat andthus not protruding into the intestinal or esophageal or gastric lumen

According to a preferred embodiment of the invention herein disclosed,the pharmaceutical composition in form of emulsion or microemulsion isin liquid phase both at room temperature (i.e. about 20-25° C.) and atbody temperature (i.e. about 37° C.). According to another preferredembodiment, said composition has a viscosity below about 150 cP(centipoises), more preferably below about 100 cP (centipoises), muchmore preferably below about 50 cP (centipoises). According to anotherpreferred embodiment, said composition has a viscosity below about 20 cP(centipoises), preferably below about 10 cP (centipoises).

According to the invention, said viscosity is measured at about 25° C.,at about 30° C. and/or at about 37° C., preferably using a BrookfieldLVDV-III Ultra Programmable Rheometer™ equipped with a Brookfield SmallSample Adapter™ device and using Brookfield™ spindle N. 31.Alternatively, said viscosity is measured using a Brookfield LVDV-IIIUltra Programmable Rheometer™ equipped with a Brookfield Enhanced ULAdapter™ device and using Brookfield™ spindle N. 00.

In a preferred embodiment, the viscosity of said pharmaceuticalcomposition in form of emulsion or microemulsion, measured at 25° C., isbelow 150 cP, preferably below 100 cP, more preferably below 50 cP. In apreferred embodiment, the viscosity of said pharmaceutical compositionin form of emulsion or microemulsion, measured at 30° C., is below 150cP, preferably below 100 cP, more preferably below 50 cP. In a preferredembodiment, the viscosity of said pharmaceutical composition in form ofemulsion or microemulsion, measured at 37° C., is below 150 cP,preferably below 100 cP, more preferably below 50 cP. In a morepreferred embodiment, the viscosity of said pharmaceutical compositionin form of emulsion or microemulsion, measured at 25° C., at 30° C.and/or at 37° C. is below 20 cP, preferably below 10 cP.

The presence of at least one dye into the cushion helps the endoscopistto visualize the structures beneath the mucosa (e.g. the submucosallayer and the external muscular wall), thereby lowering the risk thatthe endoscopist, performing the resection procedure, may cause damagesto said structures: as a matter of facts, the dye make him able todistinguish between the cushion cavity and the mucosal basement. Theremoval of the lesion from the mucosal surface generates a hole into thebasement that has to be healed and the presence, into the pharmaceuticalcompositions according to the invention herein disclosed, of an agentcharacterized by trophic activity on the epithelial cells of thegastrointestinal mucosa has the aim of accelerating the healing of themucosal wound. The persistency of the cushion generated by the injectedvolume of the pharmaceutical composition in form of emulsion ormicroemulsion according to the invention herein disclosed islong-lasting enough to allow the endoscopic resection procedure to beperformed without the need to re-inject said composition every couple ofminutes, as it generally happens when normal saline solution is used.

In view of the above, a first advantage provided by the composition inform of emulsion or microemulsion of the invention is to ensure themanually administration at room temperature (20-25° C.) without the needof cooling the composition and/or the endoscopic injection needle.

A second advantage of the composition of the invention is to avoid anyrisk to have unwanted gelation into the endoscopic injection needle,while the composition is administered during the endoscopic procedure.

A further advantage of the composition of the invention is the abilityto provide a cushion high and/or long-lasting enough to allow a safecompletion of the endoscopic resection procedure, such as polypectomy,EMR and/or ESD.

A further advantage is the possibility to add at least one dye,obtaining the improvement of the visibility of the submucosal layer tothe operator with the consequent improvement of the safety and thereduction of the risk of damaging the structures beneath the mucosa.

A further advantage is the possibility to add at least one trophicagent, obtaining the improvement of the wound healing of the mucosa,with the promotion of the cellular growth and related differentiation.

DEFINITIONS

References in the specification to “one embodiment”, “an embodiment” andsimilar indicate that the described embodiment may include a particularaspect, feature, structure or characteristic. Moreover, such phrasesmay, but do not necessarily, refer to the same embodiment referred to inother portions of the specification. Further, when a particular aspect,feature, structure or characteristic is described in connection with anembodiment, it is within knowledge of a person skilled in the art toaffect or connect said aspect, feature, structure or characteristic withother embodiments, whether or not explicitly described.

The terms “comprising”, “having”, “including” and “containing” are to beconstrued as open-ended terms (i.e. meaning “including, but not limitedto”) and are to be considered as providing support also for terms as“consist essentially of”, “consisting essentially of”, “consist of” or“consisting of”.

The terms “consist essentially of”, “consisting essentially of” are tobe construed as a semi-closed terms, meaning that no other ingredientswhich materially affects the basic and novel characteristics (andoptionally physiologically acceptable excipients and/or adjuvants) ofthe invention are included.

The terms “consists of”, “consisting of” are to be construed as a closedterm.

The singular forms “a”, “an” and “the” include plural references unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for theuse of exclusive terminology, such as “solely”, “only”, and the like, inconnection with the recitation of claims elements or use of a “negative”limitation.

The term “and/or” means anyone of the items, any combination of theitems, or all the items with which this term is associated.

Unless indicated otherwise herein, the term “about” is intended toinclude values, e.g. weight percentages, proximate to the recited rangethat are equivalent in terms of the functionality of the individualingredient, the composition, or the embodiment.

A person skilled in the art will recognize that, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited rangeincludes each specific value, integer, decimal, or identity within therange.

A person skilled in the art will recognize that where members aregrouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of anyone or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby anyone or more of the recited elements, species, or embodiments,may be excluded from such categories or embodiments, for example, asused in an explicit negative limitation.

As well known in the art, the term “Emulsion” refers to a heterogeneouspreparation composed of two immiscible liquids (by convention describedas oil and water), one of which is dispersed as fine droplets uniformlythroughout the other. The phase present as small droplets is called thedisperse, dispersed, or internal phase and the supporting liquid isknown as the continuous or external phase. Emulsions are convenientlyclassified as oil-in-water (o/w) or water-in-oil (w/o), depending onwhether the continuous phase is aqueous or oily.

“Microemulsions” are thermodynamically stable, transparent (ortranslucent) dispersions of oil and water that are stabilized by aninterfacial film of surfactant molecules. The surfactant may be pure, amixture, or combined with a co-surfactant such as a medium-chainalcohol. Microemulsions are readily distinguished from normal emulsionsby their transparency, their low viscosity, and more fundamentally theirthermodynamic stability and ability to form spontaneously. The dividingline, however, between the size of a swollen micelle (˜10-140 nm) and afine emulsion droplet (˜100-600 nm) is not well defined, althoughmicroemulsions are very labile systems and a microemulsion droplet maydisappear within a fraction of a second whilst another droplet formsspontaneously elsewhere in the system. The above definitions of“emulsion” and “microemulsion” were taken from Gillian M. Eccleston“Emulsion and Microemulsion” Encyclopedia of Pharmaceutical Technology,2007, 3^(rd) edition, Informa Healthcare.

The term “endoscopic mucosal resection” (EMR) refers to an endoscopictechnique developed for removal of sessile or flat neoplasms confined tothe superficial layers (mucosa and submucosa) of the GI tract.

The term “endoscopic mucosal dissection” (ESD) refers to an endoscopictechnique developed specifically for removing larger lesions.

“Endoscopic injection needles”, known also under the names of “injectionneedles” or “injection needle catheters” or “endoscopic injection needlecatheters”, are devices which can be long up to about 230 cm and whichinclude a relatively long catheter within which an inner injection tubehaving a distal injection needle is slideably disposed. Generally, aproximal actuating handle is coupled to the catheter and the injectiontube for moving one relative to the other when necessary. The needle isgenerally retractable. Fluid access to the injection tube is typicallyprovided via a luer connector on the handle. Endoscopic injection needledevices are typically delivered to the injection site through theworking channel of the endoscope. In order to protect the lumen of theendoscope working channel from damage, the handle of the infusion needledevice is manipulated to withdraw the distal injection needle into thelumen of the catheter before inserting the device into the endoscope.This is important to prevent exposure of the sharp point of theinjection needle as the device is moved through the lumen of theendoscope. When the distal end of the endoscopic injection needle deviceis located at the injection site, its handle is again manipulated tomove the injection needle distally out of the lumen of the catheter.When advanced to the most distal position, the exposed portion of theinjection needle is approximately 4-6 mm in length.

“In (or under) laboratory test conditions” or “in laboratory conditions”or “in laboratory tests”, as used herein, refer to in-vitro conditions,such as methods, equipment and instruments commonly used in laboratorytests to perform a physical-chemical characterisation of a composition.The term refers to methods, equipment and instruments used and performedin laboratory. By way of example, the viscosity test or the test of theclimatic chamber, described in the Examples 6 and 7 reported hereinafterand used to verify whether a composition is in liquid phase or in gelphase, are tests performed in laboratory, thus they are performed in“laboratory test conditions”.

“Up to 40° C.” or “temperature up to 40° C.” refer to any temperaturecomprised between 5° C. and 40° C., preferably about 5° C., about 20°C., about 25° C. about 30° C. and/or 37° C.

“Body temperature” refers to the level of heat produced and sustained bythe body processes. Heat is generated within the body through metabolismof nutrients and lost from the body surface through radiation,convection, and evaporation of perspiration. Heat production and lossare regulated and controlled in the hypothalamus and brainstem. Normaladult body temperature, as measured orally, is 37° C., even thoughlittle variations are normally recorded throughout the day.

“Room temperature” (RT) is generally defined as the ambient airtemperature in whatever environment being used for a given procedure.More specifically, it is defined as 20-25° C., as some ambienttemperatures, by nature, do not fall within this range. Generally,protocols calling for steps to be performed at RT require thattemperatures do not fall below 18° C., and do not exceed 27° C.

“Critical Gelation Concentration” (CGC), for a composition containing aninverse thermosensitive polymer represents the concentration of saidpolymer above which said composition is able to transition from a liquidphase to a gel phase in response to the raise in temperature.

“Critical Gelation Temperature” (CGT) represents the temperature abovewhich a composition containing an inverse thermosensitive polymer at aconcentration equal to or above the critical gelation concentrationtransitions from a liquid phase to a gel phase.

“Lugol's solution”: is a solution of elemental iodine and potassiumiodide in water.

The “viscosity” defines the resistance of a liquid or semisolid againstflow. The flow of liquids or semisolids is described by viscosity, or,more precisely, by shear viscosity q. The shear viscosity of a fluidexpresses its resistance to shearing flows, where adjacent layers moveparallel to each other with different speeds. Common units ofmeasurement of viscosity are the pascal-second (Pa·s), the poise (P) and“cP” centipoises. 1 poise (P) corresponds to 0.1 pascal-second (Pa·s); 1centipoise (cP) corresponds to 1 millipascal-second (mPa·s).

“Percentage by weight with respect to the weight of the composition(w/w)” and “Percentage by weight with respect to the volume of thecomposition (w/v)”: define the percentage amount of a component orsubstance in the composition. Considering that the density of thecomposition in form of emulsion or microemulsion is equivalent to thedensity of the water (1.0 g/mL), the percentage by weight with respectto the weight of the composition (w/w) is considered equivalent topercentage by weight with respect to the volume of the composition(w/v). For the purpose of the present invention, the two definitions aretherefore interchangeable.

PEG: polyethylene glycol.

The following examples are included for purpose of illustration ofcertain aspects and embodiments of the invention, and are not intendedto limit the invention.

EXAMPLES Example 1 Emulsion

Component g/100 g Methylene blue 0.0010 Sodium chloride 0.9000 Poloxamer188 10.0000 Soybean oil 0.1600 Glycerol 0.0050 Egg lecithin 0.0240Sodium oleate 0.0006 Water for injection q.s. 100.0 g

The manufacture of the composition is described hereinafter (for 10.00Kg of final composition):

a) In a suitable vessel provided with a stirrer, 8600 mL of water forinjection are loaded; then, 90.00 g of sodium chloride are added. Themixture is kept under stirring until a complete dissolution is achieved.The obtained solution is cooled at a temperature ranging between 5° C.and 10° C.; then, 1000.00 g of poloxamer 188 are added under stirring.The mixture is kept under stirring until a complete dissolution isachieved.

b) In a suitable vessel provided with a stirrer, about 181 mL of waterfor injection are loaded; the temperature is raised at 80° C. 2.40 g ofegg lecithin, 0.50 g of glycerol and 0.06 g of sodium oleate are addedunder stirring. The stirrer is operated until complete homogenization;then, 16.00 g of soybean oil are added. The mixture is kept at T=80° C.under stirring until an homogeneous emulsion is obtained. The emulsionis then cooled at a temperature below 30° C.

c) The emulsion obtained in step b) is added to the mixture obtained instep a) under stirring. Then, 0.10 g of methylene blue are added understirring. The mixture is kept under stirring until homogeneity.

d) The pH of the mixture of step c) is measured and it is brought, ifnecessary, within the range 5.0-7.0.

e) The mixture is brought to a final weight of 10.00 Kg by adding waterfor injection.

f) The final composition is filtered through a 0.45 μm filter and ispacked in 20 mL vials capped with rubber caps and aluminum rings. Thevials are sterilized at 121° C. for 20 minutes.

Example 2 Emulsion

Component g/100 g Methylene blue 0.0010 Sodium chloride 0.9000 Poloxamer407 9.0000 Soybean oil 0.1600 Glycerol 0.0050 Egg lecithin 0.0240 Sodiumoleate 0.0006 Water for injection q.s. 100.0 g

The composition was obtained by a process similar to that described inExample 1.

Example 3 Emulsion

Component g/100 g Methylene blue 0.0010 Sodium chloride 0.9000 Poloxamer188 10.0000 Soybean oil 0.0800 Glycerol 0.0025 Egg lecithin 0.0120Sodium oleate 0.0003 Water for injection q.s. 100.0 g

The composition was obtained by a process similar to that described inExample 1.

Example 4 Emulsion

Component g/100 g Methylene blue 0.0010 Sodium chloride 0.9000 Poloxamer407 9.0000 Soybean oil 0.0800 Glycerol 0.0025 Egg lecithin 0.0120 Sodiumoleate 0.0003 Water for injection q.s. 100.0 g

The composition was obtained by a process similar to that described inExample 1.

Example 5 Emulsion

Component g/100 g Methylene blue 0.0010 g Sodium chloride 0.9000 gL-Glutamic acid 1.0000 g Poloxamer 188 10.000 g Soybean oil 0.1600 gGlycerol 0.0050 g Egg lecithin 0.0240 g Sodium oleate 0.0006 g Sodiumhydroxide q.s. to bring the pH within 5.0 and 7.0 Water for injectionq.s. to 100.0 g

The manufacture of the composition is described hereinafter (for 10.00Kg of final composition):

a) In a suitable vessel provided with a stirrer, 8600 mL of water forinjection are loaded; then, 90.00 g of sodium chloride are added. Themixture is kept under stirring until a complete dissolution is achieved.The obtained solution is cooled at a temperature ranging between 5° C.and 10° C.; then, 1000.00 g of poloxamer 188 are added under stirring.The mixture is kept under stirring until a complete dissolution isachieved.

b) In a suitable vessel provided with a stirrer, about 181 mL of waterfor injection are loaded; the temperature is raised at 80° C. 2.40 g ofegg lecithin, 0.50 g of glycerol and 0.06 g of sodium oleate are addedunder stirring. The stirrer is operated until a complete homogenization;then, 16.00 g of soybean oil are added. The mixture is kept at T=80° C.under stirring until an homogeneous emulsion is obtained. The emulsionis then cooled at a temperature below 30° C.

c) The emulsion obtained in step b) is added to the mixture obtained instep a) under stirring. Then, 0.10 g of methylene blue and 100.00 g ofL-glutamic acid are added under stirring. The mixture is kept understirring until homogeneity.

d) The pH of the mixture of step c) is measured and it is brought withinthe range 5.0-7.0 by adding 10% NaOH in water for injection.

e) The mixture is brought to a final weight of 10.00 Kg by adding waterfor injection.

f) The final composition is filtered through a 0.45 μm filter and ispacked in 20 mL vials capped with rubber caps and aluminum rings. Thevials are sterilized at 121° C. for 20 minutes.

Example 6 Viscosity Measurement in Laboratory Test

The viscosities of the pharmaceutical compositions according to Examples1 to 4 were measured in laboratory conditions at three differenttemperatures: T=25° C., T=30° C. and T=37° C. The measurements wereperformed using a Brookfield LVDV-III Ultra Programmable Rheometer™equipped with a Brookfield Small Sample Adapter™ device. The BrookfieldSmall Sample Adapter™ comprised a sample chamber which fitted into awater jacket so that precise temperature control was achieved by meansof a circulating thermostating water bath. For the measurements, twodifferent spindles were used, depending upon the viscosity value: forlow viscosity values (registered at T=25° C., 30° C. and 37° C. forcompositions according to example 1 to 4 and at T=25° C. and 30° C. forthe reference), Brookfield™ spindle N. 31 was used; for high viscosityvalues (registered at T=37° C. for the reference), Brookfield™ spindleN. 25 was used.

A solution of poloxamer 407 in normal saline was used as reference. Thereference was prepared dissolving poloxamer 407 in normal saline toobtain a final concentration of poloxamer 407 equal to its criticalgelation concentration (about 15% by weight with respect to the totalweight of the solution). The composition of the reference solution ishereinafter reported:

Component g/100 g Sodium chloride 0.9000 Poloxamer 407 15.0000 Water forinjection q.s. 100.0 g

The viscosities of the compositions according to Examples 1 to 4 arereported in the table below with respect to the reference solution:

Viscosity (cP) Viscosity Viscosity Viscosity Composition at 25° C. at30° C. at 37° C. Reference (solution) 60 88 1044 Composition of Example1 5.70 5.45 4.95 Composition of Example 2 6.45 6.80 6.30 Composition ofExample 3 5.70 5.60 5.00 Composition of Example 4 6.70 6.75 6.25

The reference solution showed a gel-forming ability upon heating from25° C. to body temperature (i.e. 37° C.) in laboratory conditions,passing from a liquid state having a viscosity of about 60 cP to a gelstate, having a viscosity of 1044 cP. The pharmaceutical compositionsaccording to Examples 1 to 4 did not show any gel-forming ability, sincetheir viscosities remained quite constant upon heating from 25° C. tobody temperature (i.e. 37° C.).

Example 7 Phase Characterization by Means of a Climatic Chamber Test

In order to characterize whether a composition in form of emulsion ormicroemulsion of the invention is in liquid phase or in gel phase, aclimatic chamber test was performed in addition to the viscosity testdescribed in Example 6. The pharmaceutical compositions according toExamples 1 to 4 and the reference solution described in Example 6(poloxamer 407 15% in normal saline) were packed_in sealed vials, whichwere then placed into a climatic chamber thermostated at 40° C. Aftertwo hours, the phase (liquid or gel) of said compositions was easilychecked turning upside down the vials: in the case of the compositionsaccording to Examples 1 to 4, there was a flow of liquid while the vialwas being turned upside down; on the contrary, in the case of thereference solution (poloxamer 407 15% in normal saline), there was noflow of liquid into the vial, and the composition in gel phase remainedatop the vial.

Example 8 Injection of the Composition According to Example 1 into theSubmucosal Layer of an Ex Vivo Porcine Stomach

An ex vivo porcine stomach was placed into a water-bath maintained at37.0° C.±0.5° C. by means of a calibrated thermostat. Once the stomachreached the desired temperature (37° C.±0.5° C.), it was then placed onan examination couch. The composition according to Example 1 wasinjected into the submucosal layer of the stomach by means of anendoscopic injection needle; the injected volume was 5.0 mL+0.5 mL, inorder to create a visually adequate submucosal elevation. Two injectionswere performed; in both cases, the generated submucosal cushions (FIGS.1 and 2) were able to elevate the mucosal wall in a way suitable toallow a typical resection of polyps by means of a snare or of anelectroscalpel, in accordance to typical endoscopic resection proceduressuch as EMR and ESD. One of the cushions was cut by means of a scalpelimmediately after the injection; after the cut, the composition seemedto have provided a viscous product into the submucosal layer which had agood consistency (FIG. 3). A specimen of the mucosa was resected andvisually examined: the product formed by the composition according toExample 1 remained attached to the piece excised (FIG. 4). The othercushion was held in place for 15 minutes from the injection beforecutting. During this time, the cushion did not show any change in shapeand in height (FIG. 5). After 15 minutes, the second cushion was cut ina way similar to the first cushion (FIG. 6). The visual examination ofthe specimen after cutting revealed the presence of a viscous productinto the submucosal layer which had a consistency similar to thatobtained in the first cushion (FIG. 7). The test revealed that thecomposition according Example 1, which was not able to transition from aliquid phase to a gel phase upon heating from 25° C. to body temperature(i.e. 37° C.) in laboratory test conditions (as reported in Examples 6and 7), was contrarily able to generate a high, long-lasting submucosalcushion once injected into the submucosal layer of a porcine stomach.The cutting of such a cushion revealed that the composition according toExample 1 had surprisingly formed a viscous product into the submucosallayer; after the removal of a specimen of mucosa from the cushion, sucha product remained attached to said specimen for 10 minutes.

Example 9 Microemulsion

Component g/100 ml Methylene blue 0.0010 Sodium chloride 0.5000Poloxamer 188 10.0000 Soybean oil 0.0050 PEG-15 hydroxystearate 0.1000Water for injection q.s. to 100.0 ml

The manufacture of the composition is described hereinafter.

a) In a suitable vessel provided with a stirrer, the lipophilic compoundand the non-ionic surfactant are loaded and mixed; then a suitableamount of warm water for injection is poured into the oily phase understirring. The mixture is maintained stirred and warmed until amicro-emulsion is obtained.

b) In a second tank, the remaining amount of water for injection iswarmed; then the micro-emulsion prepared at step a) is poured drop wiseunder stirring.

c) The polymer is added to the micro-emulsion of step b), and themixture is maintained under stirring until complete dissolution isachieved.

d) Sodium chloride is added under stirring until complete dissolution isachieved.

e) The dye is added under vigorous stirring until complete dissolutionis achieved.

f) The pH of the mixture of step e) is measured (specification:5.0-7.5).

g) The mixture is brought to final volume by adding water for injection.

h) The final composition is sterilized by sterilizing filtration thanksto the very small droplets size (below 100 nm) of the micro-emulsion;thus it is filtered through a 0.22 μm filter and is packed by asepticprocessing in vials capped with rubber caps and aluminium rings.

Example 10 Microemulsion

Component g/100 ml Methylene blue 0.0010 Sodium chloride 0.5000Poloxamer 188 10.0000 Medium chain triglycerides 0.0200 PEG-15hydroxystearate 0.0800 Water for injection q.s. 100.0 ml

The composition was obtained by a process similar to that described inExample 9.

Example 11 Microemulsion

Component g/100 ml Methylene blue 0.0010 Sodium chloride 0.5000Poloxamer 188 10.0000 Soybean oil 0.2000 Polyoxyl-35 castor oil 3.0000Water for injection q.s. to 100.0 ml

The composition was obtained by a process similar to that described inExample 9.

Example 12 Microemulsion

Component % w/v Methylene Blue 0.001 Sodium Chloride 0.440 Poloxamer 18810.000 Polyoxyl-15 Hydroxystearate 0.500 Medium chain triglycerides0.100 WFI q.s. to 100 ml

The composition was obtained by a process similar to that described inExample 9.

Example 13 Microemulsion

Component g/100 ml Methylene blue 0.0010 Sodium chloride 0.5000Poloxamer 188 10.0000 Medium chain triglycerides 1.0000 PEG-15hydroxystearate 4.0000 Water for injection q.s. to 100 ml

The composition was obtained by a process similar to that described inExample 9.

Example 14 Microemulsion

Component g/100 ml Indigo carmine 0.001 Sodium chloride 0.500 Poloxamer188 10.000 Polyoxyl-15 Hydroxystearate 0.600 Medium chain triglycerides0.100 Water for injection q.s. to 100 ml

The composition was obtained by a process similar to that described inExample 9.

Example 15 Microemulsion

Component g/100 ml Methylene Blue 0.001 Sodium chloride 0.500 Poloxamer188 10.000 Polyoxyl-15 Hydroxystearate 0.500 Medium chain triglycerides0.100 Water for injection q.s. to 100 ml

The composition was obtained by a process similar to that described inExample 9.

Example 16 Microemulsion

Component g/100 ml Sodium chloride 0.500 Poloxamer 188 10.000Polyoxyl-35-castor oil 3.000 Soybean oil 0.200 Water for injection q.s.to 100 ml

The composition was obtained by a process similar to that described inExample 9, without the step e).

Example 17 Characterisation of the Micro-Emulsion Droplet Size byDynamic Light Scattering (DLS)

The oil-in-water micro-emulsions of the present invention arethermodynamically stable, can be prepared spontaneously, and aretransparent.

The dynamic light scattering (DLS) technique was used to characterizethe micro-emulsion droplet size.

-   -   INSTRUMENT: Zetasizer Nano® ZSP from Malvern Instruments    -   SAMPLE PREPARATION: None, sample not diluted    -   SETTING UP MEASUREMENT:    -   Measurement type: size    -   Sample:        -   Material: No setting (The material optical properties are            not needed for intensity-based distribution)        -   Dispersant: water with bulk viscosity at 25° C.        -   General options: use dispersant viscosity as sample            viscosity        -   Temperature: 25° C. with 60 sec of equilibration time        -   Cell: disposable cuvettes DTS0012    -   MEASUREMENT:        -   Measurement angle: 173° backscatter (NIBS default)        -   Measurement duration: Automatic        -   Number of measurement: at least 3    -   Instructions: no one    -   Advanced:        -   Extend duration for large particles: No        -   Positioning method: Seek for optimum position        -   Automatic Attenuation Selection: Yes

Hereinafter the DLS analyses of the micro-emulsion of Example 15 areshown.

Two samples were withdrawn at the end of step a) and d), and were thenanalyzed using the instrument parameters reported above.

In the following table A the results of DLS analysis on sample from stepa) are reported. The relevant graph is shown in FIG. 8.

TABLE A Size % St. Dev. (d · nm): Intensity: (d · nm) Z-Average (d · nm)14.61 Peak 1 15.41 100.0 3.790 Pdl 0.036 Peak 2 0.000 0.0 0.000Intercept 0.949 Peak 3 0.000 0.0 0.000

The results show the distribution of monodisperse particles with aZ-Average around 14 nm and a polydispersity index extremely low.

In the following table B the results of DLS analysis on sample from stepd) are reported. The relevant graph is shown in FIG. 9.

TABLE B Size % St. Dev. (d · nm): Intensity: (d · nm) Z-Average (d · nm)14.16 Peak 1 18.25 100.0 5.764 Pdl 0.269 Peak 2 0.000 0.0 0.000Intercept 0.960 Peak 3 0.000 0.0 0.000

The chart shows a unique distribution of particles with a Z-Averagearound 14 nm and a low polydispersity index. The measurements arereproducible with a good intercept of the correlation function (0.960).

In the following table C, a comparison between the results obtained atstep a) and step d) is reported. The relevant graph is shown in FIG. 10.

TABLE C Sample Zav (nm) PdI Sample from step a) 14.39 0.24 Sample fromstep d) 14.43 0.27

From the superposition of the particle size distributions and the twodimensional data, the two samples are equal: the small differencesbetween them are not significant and can be attributed to experimentalvariability. Thus, the two samples are equal both in terms ofdistribution and of the Z-average.

In the following table D the results of DLS analysis on sample from stepe) are reported. The relevant graph is shown in FIG. 11.

TABLE D Size % St. Dev. (d · nm): Intensity: (d · nm) Z-Average (d · nm)28.02 Peak 1 44.82 100.0 22.21 Pdl 0.303 Peak 2 0.000 0.0 0.000Intercept 0.147 Peak 3 0.000 0.0 0.000

The graph shows a single distribution of particles with a Z-Averagearound 28 nm.

The DLS analyses of the micro-emulsion on sample from step e) of theExamples 11-13 and 14 are reported in the following table E:

TABLE E EXAMPLES NUMBER Z-Average (d · nm) Pdl 11 9.61 0.20 13 15.150.11 14 27.51 0.223

The DLS analyses of the micro-emulsion on sample from step a), d) ofExample 12 are reported in the following table F

TABLE F EXAMPLES NUMBER Z-Average (d · nm) Pdl 12 after step a) 13.980.159 12 after step d) 16.68 0.305

Example 18 Cytotoxicity

The composition according to Example 15 was subjected to an in vitrocytotoxicity study on Mammal fibroblasts ATCC BalbC 3T3, according toISO 10993-5.

After 24 hours of test, the following results were obtained:

The reduction of vitality of the cells in the well of the composition ofExample 15 was 14.68%, and the composition was judged to be notcytotoxic.

Example 19 Cytotoxicity

The composition according to Example 10 was subjected to an in vitrocytotoxicity study on Mammal fibroblasts ATCC BalbC 3T3, according toISO 10993-5.

After 24 hours of test, the following results were obtained:

The reduction of vitality of the cells in the well of the composition ofExample 10 was 6.22%, and the composition was judged to be notcytotoxic.

Example 20 Testing on Ex Vivo Porcine Stomachs

During product development, the cushion forming ability of the differentprototype formulations has been evaluated using several tests on ex vivoporcine stomachs. The porcine stomach was selected as testing systembecause it is a widely accepted model of the human gastrointestinalmucosa. Moreover, in scientific literature many published works onsubmucosal injection agents describe the use of this model for assessingthe performance of the different agents in terms of height and durationof the submucosal cushion.

The efficacy of the pharmaceutical compositions according to the presentinvention was evaluated in the ex vivo test, in terms of height andduration of the submucosal cushion following the injection of a suitablevolume.

A brief description of the method is reported hereinafter.

Materials

-   -   Frozen porcine stomach    -   Plexiglass support.    -   10 ml Luer-Lock Syringe    -   Standard Endoscopic injection needle

Method

The frozen porcine stomach is thawed out and then is kept at 37° C. in athermal blanket. The stomach is cut open using a surgical scalpel, andthe internal mucosa is cleaned up using paper towels. A 10 cm×10 cmsquare portion is cut from the stomach and is fitted in the Plexiglassupport. A suitable volume of the pharmaceutical composition is injectedthrough the endoscopic injection needle into the submucosal layer of theresected square specimen of the porcine stomach When the submucosalcushion formation is completed, the needle is removed from the specimen.The height and time of permanence of the obtained submucosal cushion isevaluated by visual inspection. The cushion is monitored every 15minutes up to an hour.

Results

As depicted in FIG. 12, the submucosal cushion created after injectionof a suitable amount of the composition of Example 11 passed from aheight of 1.6 cm to 1.4 cm, thus losing only 0.2 cm over 1 hour frominjection.

The results for the compositions of examples 9, 11 and 13 are reportedin following table G:

TABLE G EXAMPLE NUMBER HEIGHT AT T 0′ HEIGHT AT T 60′ 11 (see FIG. 12)1.6 cm 1.4 cm  9 1.1 cm 1.1 cm 13 1.2 cm 1.1 cm

Example 21 Preliminary Test on In Vivo Minipig

A preliminary tolerance study on a minipig was carried out oncomposition according to Example 5.

Purpose

The purpose of the study was to investigate the tolerance of the productin minipig after gastric submucosal administration.

Methods

One male Göttingen minipig, having a weight of approximately 20 kg andan age of approximately 10 months, was used for this study. Theendoscopic procedure was performed with the use of an Electronic VideoEndocopes Fujinon EVE200 System and Upper Gastrointestinal ElectronicVideo Endocopes EG-201FP. The submucosal injection agent was deliveredby means of the endoscope using and endoscopic injection needle. Theanimal was anaesthetized prior to each endoscopic procedure. The testitem was administered (about 5 ml) by endoscopic submucosal injection,using an endoscopic injection needle (Medwork® injection needle, 230cm×2.3 mm, needle diameter 0.7 mm, Ref. N. INJI-A1-07-5-23-230). Theanimal was dosed once by submucosal injection in about 55 seconds,followed by observation for 24 hours. After administration, the mucosaof the injection site and the surrounding untreated mucosa werecontinuously examined for 25 minutes, during which the test item causedan adequate distension with a detachment between the mucosal andsubmucosal layers. This detachment was still persistent 25 minutes afterinjection; further examinations were performed at 60 minutes and at 24hours.

The subsequent overall observation of the injection site at about 60minutes after the injection, showed the persistence of evidentswellings.

At 24-hour observation period, gastric mucosa swellings were no longerpresent and gastric mucosa showed no test article related grossmacroscopic changes.

FIGS. 13, 14 and 15 show the administration of the test item, thesubmucosal cushion, and the appearance of the injection site at 24 hourspost administration.

Example 22 Rheology

The viscosity variation as function of temperature was measured on thecomposition of example 16 by using a rotational rheometer, Kinexus pro+.

The Kinexus pro + is a rotational rheometer that applies controlledshear deformation to the sample, and it is normally used in order toevaluate and study the rheological characterization (viscosity) ofcompositions, as emulsions or microemulsions.

For proceeding with the measurement, the composition of example 16 wasequipped with a cone plate CP60 −2° at controlled shear and constantstress, 0.5 Pa; the temperature range was set between 25° C. and 50° C.

As reported in the graph of FIG. 16, the rheogram viscosity versus.temperature demonstrates that the viscosity of the composition decreaseswith the increasing of the temperature.

1. A pharmaceutical composition in the form of an emulsion or a microemulsion comprising: (a) an aqueous phase; (b) an oily phase; (c) at least one surfactant; (d) at least one inverse thermosensitive polymer, and wherein said at least one inverse thermosensitive polymer is present in an amount below the critical gelation concentration (CGC), and wherein said composition remains in liquid phase up to a temperature of about 40° C. in vitro.
 2. The pharmaceutical composition according to claim 1, wherein said composition has a viscosity below about 150 cP (centipoises).
 3. The pharmaceutical composition according to claim 1, wherein said at least one inverse thermosensitive polymer is present in an amount below about 15% by weight, with respect to the weight of the composition.
 4. The pharmaceutical composition according to claim 3, wherein said at least one inverse thermosensitive polymer is present in an amount between about 2% and about 14.5% by weight, with respect to the weight of the composition.
 5. The pharmaceutical composition according to claim 4 wherein said at least one inverse thermosensitive polymer is present in an amount between about 5% and about 11% by weight, with respect to the weight of the composition.
 6. The pharmaceutical composition according to claim 1, wherein said at least one inverse thermosensitive polymer is at least one poloxamer.
 7. The pharmaceutical composition according to claim 6, wherein said at least one poloxamer is poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 407 or mixtures thereof.
 8. The pharmaceutical composition according to claim 6, wherein said at least one poloxamer is poloxamer 188, poloxamer 407 or mixtures thereof.
 9. The pharmaceutical composition according to claim 1, wherein said oily phase comprises at least one lipophilic compound.
 10. The pharmaceutical composition according to claim 9, wherein said at least one lipophilic compound is a medium-chain triglycerides, soybean oil, sesame oil or almond oil.
 11. The pharmaceutical composition according to claim 1, wherein said at least one surfactant is at least one non-ionic surfactant.
 12. The pharmaceutical composition according to claim 11, wherein said at least one non-ionic surfactant is polysorbate 80 or polyoxyl-15-hydroxystearate.
 13. The pharmaceutical composition according to claim 1, wherein said at least one surfactant is at least one ionic surfactant.
 14. The pharmaceutical composition according to claim 13, wherein said at least one ionic surfactant is egg lecithin, hydrogenated phosphatidyl choline from egg lecithin, soybean lecithin or hydrogenated soybean lecithin.
 15. The pharmaceutical composition according to claim 1, further comprising at least one co-surfactant wherein said at least one co-surfactant is propylene glycol, glycerol or sodium oleate.
 16. The pharmaceutical composition according to claim 1, further comprising at least one dye wherein said at least one dye is a vital dye, a non-vital dye or a reactive dye.
 17. The pharmaceutical composition according to claim 16, wherein said at least one dye is Lugol's solution, methylene blue, toluidine blue, crystal violet, indigo carmine, Congo red or phenol red.
 18. The pharmaceutical composition according to claim 17, wherein said at least one dye is methylene blue.
 19. A pharmaceutical composition in the form of an emulsion or a microemulsion comprising: (a) an aqueous phase; (b) an oily phase comprising medium-chain triglycerides; (c) a surfactant comprising polyoxyl-15-hydroxystearate; (d) an inverse thermosensitive polymer comprising poloxamer 188; and (e) methylene blue; wherein said poloxamer 188 is present in an amount below the critical gelation concentration (CGC), and wherein said composition remains in liquid phase up to a temperature of about 40° C. in vitro.
 20. The pharmaceutical composition according to claim 19, which further comprises sodium chloride.
 21. The pharmaceutical composition according to claim 19, wherein the composition comprises in w/v about 0.02% to about 1.0% of medium-chain triglycerides, about 0.08% to about 4% polyoxyl-15-hydroxystearate, about 5% to about 11% poloxamer 188, about 0.0005% to about 0.01% methylene blue and the remainder being water.
 22. The pharmaceutical composition according to claim 20, wherein the composition comprises in w/v about 0.02% to about 1.0% of medium-chain triglycerides, about 0.08% to about 4% polyoxyl-15-hydroxystearate, about 5% to about 11% poloxamer 188, about 0.0005% to about 0.01% methylene blue, about 0.44% to about 0.5% sodium chloride and the remainder being water. 